bioanalytics II analytical methods in clinical praxis Jan Havliš, Ph.D. Masaryk Uni, Fac Sei recommended reading Tietz fundamentals of clinical chemistry C.A. Burtis, E.R. Ashwood, D.E. Bruns, Elsevier, 2008 ^®®® Creative Commons: Attribution-Noncommercial-Share alike 3.0 Unported License lecture syllabus laboratory medicine samples - character, preparation instrumentation, integration and miniaturisation quality check and control choice of analytical method optimisation approach analytical set analytical result expression basic methods and principles colourness and its analytical use, indicator reactions protein determination and enzymatic analysis immunoanalysis analysis of nucleic acid medical microbiology determination of chosen analytes : case study: determination of ALP : albumin, barbiturates, sodium, ethanol... 2 laboratory medicine I. : analysis of body fluid component with diagnostical importance : determination of analytes and metabolites or their groups, including drug level monitoring : shelters branches dealing with laboratory diagnostics: clinical chemistry and biochemistry, haematology, medical microbiology, immunology classically traded as separate clinical disciplines research diagnostics <-----------------► therapy IFCC definition {international federation of clinical chemistry) „Clinical chemistry is an application of chemical, molecular and cellular principles and technologies with an aim to understand human health and illness, and to allow their classification. Presentation of analyses results in regard to the illness cause and health care is the core of the branch. On the interface of laboratory and clinic, transformation of such data follows into specific and general information related to patient and illness. Deepening of knowledge about health and illness through basic and applied research is the task of the branch." laboratory medicine : in EU 5 - 10 analyses per capita per annum : the world biggest producer of analytical data : touches almost all analytical branches laboratories hospital private consolidate laboratories diagnostic centres huge range of analytical activity : incomparable to any other science or industry branch 5 sample in laboratory mediane biological : invasive (blood, cerebrospinal liquid, tissues, gastro-duodenal juice) : non-invasive (saliva, urine, faeces, sputum, breath) blood blood = suspension of cellular particles in liquid (plasma) cellular particles : blood-cells (erythrocytes and leukocytes) : blood-platelet (thrombocytes) blood coagulation (off blood stream) - change of sol. fibrinogen =^> insol. fibrin bloocl coagulation> solid blood pie + blood serum blood serum - similar to plasma, without coagulation agents 6 plasma and serum mixture of inorganic and organic compounds in water native plasma - separated from blood-cells of blood in container with non-polar surface (plastic) without anticoagulants : as close as possible to what circulates in blood stream plasma - from blood in container with anticoagulants serum - separated for coagulated blood in container with polar surface without anticoagulants : preparation of plasma is faster : 20 % more is gained of plasma than serum : plasma lowers the risk of unwanted haemolysis (in serum up to lOx higher) : in serum after centrifugation unwanted secondary coagulation : serum is an artefact : no protein ELFO in plasma =^> fibrinogen co-elutes with the yglobulines : anticoagulants introduce ions into plasma, which could not be then analysed : lots of analytes mask and inhibit some plasma enzymes 7 urine 1 ' light yellow liquid produces by kidneys and excreted through urethra and urocyst contains: urea, chlorides, ions of sodium, potassium, phosphates, sulphates, creatinine and uric acid cerebrospinal liquid (liquor) clear, sparse liquid circulating between cerebral ventricle, central spinal channel and permeation gate between brain, spinal chord and their meninges contains: electrolytes and similar organic compounds (~ blood plasma, but different concentrations) e.g. glucose, proteins, lactate, pyruvate, cholesterol, enzymes, salts and certain amount of lymphocytes 8 what influences concentration of analytes in biological samples a) given influences b) variable influences given | race and sex e.g. different reference values of analytes: creatine kinases, a-amylases, granulocytes : higher for men than for women :: women have mostly lower and narrower reference intervals : increasing in order: Caucasian, Asiatic, African age e.g. different reference values of analytes for newborns, children, adolescents, adults and senior 9 biorhythms - chronobiological influences linear : changes according to age cyclic : daily (circadian) : monthly (lunar) : seasonal (seasons of year) biorhythms cause changes in analyte concentrations cyclic b. - varying concentrations: day-to-day, during the day important change of biorhythms during pregnancy variable influences diet : alimentation, starvation/abrosia (=> malnutrition) A concentration of fats, saccharides, proteins => A levels of serum ammonium and urea physical strain short-term and intense strain : consumption of ATP, i level of glucose and lactate long-term strain : t cone, of ions: sodium, potassium, calcium, phosphorus, ALP, albumin, urea, bilirubin, AST, pyruvate kinase, CK measure of change is individual and depends on circumstances altitude influence {stress of organism) : t concentration of C-reactive protein, ß2-globulin, uric acid, haemoglobin and haematocrit adaptation on high altitudes is slow and takes weeks adaptation back to low altitudes is fast, takes few days 11 common drugs Coffein : t glucose cone, unesterified fatty acids and catecholamines nicotine (cigarette) : acute and chronic change e.g. t cone, of serum fatty acids, glucose, fibrinogen, cholesterol, free glycerol, aldosteron and Cortisol, some hormones and tumour markers and heavy metals (Cd, Cu, Pb) alcohol : intensity and length of consumption makes influence steady influences: i cone, of serum glucose, metabolic acidose (ethanol^>acetaldehyde^>acetate) long-term influences: t act. of enzymes GMT, GLD, AST and ALT (intoxication of livers) chronic alcoholism: t cone, of triacylglycerols, cholesterol and some hormones 12 other common influences biological fluids - complex of inorg. and org. molecules, protein pseudo-solutions and fat-droplet emulsion compounds could be bound to proteins =^> their content in organism is dynamically changing; changes are related to their individual stability or decomposition processes (metabolism and bacteria) biological samples - potentially infective material =^> safety rules number of analytes in sample - hundreds + their derivatives with variable content and changeable photo- and thermostability = biological matrix influence of collection and transport - so-called preanalyticalphase necessity of fast transport and storage in cold or conservation 13 influence of medication patients samples - drug interferences results are biased or disable completely conduct of analyses information on interferences - estimated empirically : influences of individual drugs (common) : influences of drug combinations (almost unknown) : blood collected on an empty stomach after drug drop for 24 - 72 h : necessity of knowledge of drugs used by patient analytical drug interference - observed by IFCC, known analytical interferences in databank standard (normalised) operational approach (SOA) - analysis of composite sera of donors or patients enriched by known amount of respective drug : analysis result is statistically evaluated preanalytical phase collection, transport and storage of sample processes and operations with analysed material sample u n til I analysis i j biological material collection and transport analytes have limited time-stability : are metabolised, thermolabile or photolabile stability - storage period, when under defined conditions the initial analyte content is not changed; concentration or activity it is expressed as time, during which initial content of analyte is not changed more than 1.5-time more than reference interval with 95% probability 15 .*.*** collection rules influence of patient state posture - cone, of high molecular compounds are lower when collected in recline and higher in about 15 % when standing physical strain - A concentration of compounds involved in energetic metabolism, it comes to thickening of macromolecular compounds, the activity of AST and CK enzymes is increased, increases level of creatinine, decreases level of thyroxin collection in a sit-down and at least after 30 min of rest contraction of arm by elastic bandage and after disinfection oft puncture location bandage must be quickly released - freely flowing blood \s collected before puncture, patient with bandage should not exercise too long slow release of bandage and too intense arm exercise => important influence on levels oft some serum analytes: t cone. Na+ and Ca(II), haemoglobin, cholesterol, ALP, proteins, bilirubin and some enzymes; i concentration of glucose, creatinine, phosphate... 16 venous blood collection morning on empty stomach last light food at ca 6:00 pm, and then next day morning only a small amount water or tea without sugar drop medication at least for 24 - 72 hours : most of analyses (except for haematology) is done using serum : after blood collection and before separation of serum off blood-clot it is necessary to wait at least 30 minutes, what is a period needed for coagulation process : using coagulations accelerators, only 10 minutes are enough capillary blood puncture by lancet into finger, ear lobe or heel (children) : considerate : dropping or drain (by micropipette or capillary) 1-3 drops of blood : immediate analysis (determination of glucose on diagnostic strip) : transport in plastic microtube with anticoagulant and anti-glycolytic agent anticoaqulantia / anticoagulants compounds able to complexate ions of endogenous calcium in sample and thus prevent process of blood coagulation : sodium or potassium salts of citric or oxalic acid, or EDTA : heparin, accelerator antithrombin III (inhibitor of blood coagulation) : hirudin (anticoagulans of leech Hirudo medicinalis L) in haematologic analyses and tests, it is on the other side important to re-calcify uncoagulable blood and thus re-new coagulation abilities by adding of surplus of calcium salt to saturate anticoagulants 18 collection containers open system - open tube closed system - evacuated container or test-tube with rubber plug, or special injection syringe serving for sample collection and in parallel also as centrifugation tube contains - anticoagulantia or compounds speeding-up blood coagulation (gelatine, aprotinin, polystyrene spheres etc.) glucose determination - special doses with agents suppressing glycolysis in combination with anticoagulants anticoagulants are mostly salts and introduce into biological samples certain ions, which ions could not be determined in samples handled this way 19 security plug HEMOGARD sterile evacuated tube VACUTAINER© - multiple collection needle security valve predefined vacuum special separation gel - separates serum or plasma of blood coagulum or blood-cells after centrifugation; there is no need for fast separation of serum/plasma from rest of the blood containers for blood-samples collection are resolved by colour for easier manipulation; according to respective ISO norm red - clean; golden - gel for centrifugation grey - glucose (NaF, K2(ox)), green - heparin violet - EDTA, blue - citrate... 20 disposables - class of tools; closed containers and other plastic one-off tools for collection, transport, centrifugation, dosing and storing of other body fluids in sterile design are in-between them others: automatic pipettor extension, containers and test-tubes for urine samples, plastic ELISA plates, plates for determination of blood groups etc. haemolysis degradation of erythrocytes =^> changes quality of collected blood : proceeding of collected blood to serum or plasma optimally till 30 minutes, at latest till 1 hour after collection manifestation - t cone, of potassium and chlorides, t activity of enzymes ALT, i glucose appearance - normal serum and plasma - yellowish and transparent : haemolysis =^> red (release of haemoglobin) : milky cloud- emulgated fat droplets - chylous (lipaemic) serum haemolytic and chylous serum or plasma are for most analyses impropriate 21 glycolysis content of glucose in blood rapidly decreases after collection - blood-cells still live serum/plasma must be stored at +4 °C effective, but complicates transport temperature 15 - 25 °C content of glucose goes down in a day to ~30 %, in 2 days to 6 % temperature +4 °C content of glucose goes down in a day to ~80 %, in 2 days to 32 % biochemical nature of glycolysis - catabolism of glucose : aerobic glycolysis - C02 and water are final products : anaerobic glycolysis - lactate is final product : pentose cycle - direct oxidation of glucose; hexose =^> pentose 22 suppression of glycolysis : suppression of function of important catabolic enzymes glyceraldehyde-3-phosphate dehydrogenase - dehydrogenation of glyceraldehyde-3-phosphate to l,3-bis(phospho)glycerate : inhibited by monoiodacetic acid\ca 0.5 mg per ml of blood sample) enolase - transformation of 2-phosphoglycerate to phosphoenolpyruvate metalloprotein - Mg(II) in active centre : inhibited by fluorides in combination with endogenous phosphates {ca 2 mg/ml of KF or NaF; blood could be stored up to 24 h at room temperature) hexokinase - phosphorylation of glucose : inhibited by mannose in surplus (competitive substrate, 15 mmol mannose, stability of blood up to 12 h at room temperature) contraindication: no use of hexokinase method for glucose determination : inhibited by fluorides in combination of anticoagulantium (EDTA; per 1 ml of blood 1.6 mg Na2EDTA and 2 mg KF) contraindication: EDTA, KF interferes or inhibits determination of some analytes (e.g. metalloproteins) 23 urine one-shot collection (morning urine) or collected urine {12 or 24 hours) : catheterization hygiene of collection - bacterial contamination one-shot collection : basic analysis and urine sedimentation : analysis at latest till 2 hours after collection collected urine : determination of some ions, urea, glucose, microalbuminuria, creatinine and creatinine clearance : determination of some hormones (17-ketosteroides, 5-hydroxyindolacetic a. and vanillyl mandelic a.) - conservation by diluted HCl : quantitative analysis of 24 h urine - relation of analyte content to its daily excretion in urine; volume elimination conservation agents : 5 ml 10 % thymole in 2-propanol per I of urine - for most of analyses : sodium azide, 10 mmol/l urine - glucose, urea, uric acid, Na, Ca, oxalates, citrates : 25 ml HCl solution 6 mol/l for dU - 5-hydroxyindolacetic a., Ca, Mg, P, catecholamines : sodium carbonate, 2q/l urea - porphyrines, urobilinogen : benzoic acid, 10 mq na dU - glucose : adjustment of urine pH over 8 - uric acid L cerebrospinal liquor lumbal punct ion determination of proteins, glucose, chlorides etc. except for neurological investigations, routine analytical methods are used duodenal juice probe of duodenum analyses of digestion enzymes (trypsin), acids, bile acids and colorants fast changing sample, must be therefore specially collected into containers cooled with ice and immediately analysed 26 faeces, sputum, fester, saliva, sweat, sperm, mucous membrane smear and samples of organs and tissues sputum, sweat - not subject of routine analyses in clinical laboratories, usually in microbiology, histochemistry etc. faeces - i.e. occult bleeding (blood in faeces) saliva - analysis of drugs of abuse (alcohol etc.) and some steroids 27 sample preparation : whole blood centrifugation, deproteination, mineralisation, preconcentration centrifugation separation of sediment off supernatant conditions - relative centrifugation force (RCF), time and temperature RCF - how many times is the centrifugation acceleration higher at bottom then gravitational acceleration (g) intense centrifugation leads to unwanted haemolysis of blood 28 deproteination deproteination - necessary for determination of some analytes analyte : in supernatant- some ions or substrates : in sediment- organic phosphor, total protein in strong lipaemic sera, protein nitrogen by Kjeldahl method etc. deproteination techniques physical - centrifugation, ultracentrifugation, adsorption and denaturation by heat time consuming, designed for special cases chemical - immunoprecipitation, dehydration or salting-out fast 29 dehydration often used fractionation of proteins or for special analytical cases strongly depends on pH - proteins have as positive as negative charges : in acidic media - cations, in alkali media - anions : isoelectric point (pi) - specific pH at which protein is electroneutral :: at isoelectric point proteins are labile and do easily precipitate dehydration: using organic solvents or salting-out competition of protein with precipitant for water =^> takes some water off protein =^> protein is precipitated : methanol, ethanol, acetone : done at pi dehydration of proteins is mostly reversible process 30 salting-out usual precipitation of proteins in form of insoluble salts precipitants: : anionic (trichloroacetate, Perchlorate, picrate, thungstate, molybdenate, sulphosalicylate, metaphosphate) : cationic (zinc, mercury, cadmium, uranium, thorium, iron, copper and lead) mineralisation special cases only mineralisation in a dry way for determination of C, H and N by elemental analysis or determination of metal in biological/organic matrix by means of atomic absorption spectrophotometry 31 mineralisation in a wet way for determination of organic phosphorus, determination of total protein nitrogen by means Kjeldahl method kieldahlisation in clinical chemistry - mixture contains cone, sulphuric acid with different salts, e.g. K2S04 and CuS04, HgS04 and Se02 preconcentration preconcentration of trace, otherwise not determinable, amounts of analytes (proteins) methods dialysis, ultrafiltration or separation on column : not carried out in common clinical laboratories 32 instrumentation analysers; organisation, integration and analysis miniaturisation _____________________________________________________________________________________________________________________________________________________________________________i history important change in last 40 years manual plant => automatic analysers collection - blood is using injection needle put into open glass test-tube : it is either un-closed, or in better case closed with cork or rubber plug in laboratory - grumous blood mixed by glass stick, centrifuged and serum over precipitate is drained by Pasteur pipette, carry-over to other tube for analysis : samples and prepared agents are gauged into reaction tubes, and let proceed respective chemical reaction, recast reaction mixture into cuvette of photometer, measure absorbance and carry-on with analyte content calculation : determination of one analyte - 50 to 500 pi of serum and other 1.5 - 2 ml of agents comparison: around r. 1930 was volume of reaction mixture for determination of alkalic phosphatase through inorganic phosphate several millilitres and incubation lasted ca 48 hours 33 flow-through (50's) => centrifugal =^> tracked (70's) =^> revolver multi-channel analysis - parallel analyses multi-component analysis - series of analyses in one sample flow-through a. glass capillaries, samples separated by air bubbles : multi-channel analysis high accuracy and consistency of analyses centrifugal a, rotor with pits arranged radially from centre two reaction spots separated by elevated partition outer pit - photometric cuvette, transparent window perpendicular to rotor : dosing of sample and agents by centrifugation; sample fusion with agents :: recast into measurement cell rotor - 28 positions for samples, standards and controls, rinsable ~^J^= : batch analyses (one-after-one method measurements) disadvantages: not selective (no random access analyses) tracked a, copies reactions in test-tube : continuous belts placed with lines of tubes in water bath : linear movable doser pipettes serum and fixed doser individual agents : after reaction completion tube content was taken up into measuring cuvette : test-tubes are then rinsed, dried and used again for analyses :: verbatim single file of „marching tubes" disadvantage: high consumption of sample and agents revolver a. wheel of sample holders, agents and reaction cuvettes with doser system : processor controlled : absorbance measurement 340 - 600 nm by commutable filters (5 - 8) small, middle or big : average hour performance reached (analyses per hour) :: performance goes from hundreds up to thousands per hour method pallet, without re-programming: 15 - 50 special analysers - chosen groups of analytes according to medical demands : dangerous drug monitoring, urine analysis, hormone determination, oncomarkers determination, coagulation haematologic instruments (blood particles counter etc.) urgent (acute) investigations - special small a. analysers - dry chemistry dry chemistry - agents in dry state plates or strips - carriers for reagents principles of signal measurement: colourimetry, reflectometry, potentiometry and immunochemical reactions up to 50 different analyses types analyser with plates: contains holder with plates, stand for more samples and doser sample into window on one side of plate; soaked into reaction zone =^> respective chemical reaction; response is measured in window on the other side of plate analysers with diagnostic strips: reflectometric measurement of reaction spot colour 38 allows either individual analyses or analysis of a series of analytes sample 10 pi distilled water """% s- ♦ blood reagents sample nifffm product forming ♦ 612 nM mwwn rinsing 18 min. dry measurement dry chemistry - impossibility of fast sample transport or results; small analyte volume easy operation; not suitable for high-quality screening; reference determination 39 other analysers AAS (atomic absorption spectrometry), FAS (fluorescence absorption spectrometry), centrifuges, CZE and PAGE of proteins and lipoproteins, scanners, pH-metres, osmometers, coulometers, fluorimeters, NA analysers, chromatographs, mass spectrometers... change of laboratory diagnostics to laboratory medicine => advances in instrumentation! laboratory medicine <= pathology and laboratory praxis in the beginning of 19cc (C. Bernard, R. Virchow, J. Hopkins and others) till 30's used to additionally approve diagnosis - laboratory diagnostics development after 1950 - rapid increase in number of laboratory investigations with accession of novel diagnostic method, namely with immunodiagnostics (around 1960), speed up by automation and computerisation of analyses (after 1970); and by molecular biology (after 1990) 1970 - 1990 : inter-annual increase of analyses number in ca 12 %, costs of laboratory diagnostics reached already almost 10 % of total costs of health care 41 redeployment efficiency of work - consolidation of detached sections (haematology, clinical biochemistry, immunology, partially microbiology) =^> => consolidate laboratories; faster and cheaper complex service bed-side monitoring fast analysis at bed of patient by staff of clinic acute analyses in situ - so-called point-of-care testing (POCT) clinical chemistry of acute states investigation of so-called internal state of patient: blood pH, p02, pC02, ions of sodium, potassium, chlorides and haemoglobin or haematocrit patient self-monitoring (home diagnostics) analysis or investigation can patient conduct at home alone check of actual state, medication dosing, or signal to visit doctor miniaturisation and automation - robotised complexes, so-called diagnostic centres 42 centralisation consolidated laboratories simultaneous biochemical, haematological, immunochemical analyses, detection of some target NA sequences + microbiological analyses concentration of analyses originally from isolated laboratories of clinical biochemistry, haematology, microbiology, immunochemistry and so on, into a complex, which is able to provide investigation of biological samples on one place of main laboratory faster and cheaper, across traditional branch division p 43 diagnostic centres : complex analyses pallet : high degree of automation :: localisation of samples with patient bar codes on cart moving on a trajectory around mutually compatible analysers, fully automated sample gauge, analysis conduct and release finding : modular connection of compatible analysers : controlled complex : analyses practically without human touch samples of patients in disposable collection containers with bar code, which includes requested analyses + identification marks sample division into tubes as per requested analyses : samples are automatically moved between analysers : cumulation of diagnoses and patient database 44 diagnostic center organisation : core laboratory :: ca. 75 % of agency :: analytical chemistry, haematology, toxicology, immunology, urine analyses : microbiology :: ca. 20 % of agency :: blood and urine sample cultivation, serology : transfusion services :: ca. 5 % of agency :: blood-typing and pre-tranfusion cross-matching tests small, specialised laboratories acute analyses, analyses for consultation centres (diabetology, urology, toxicology etc.), where is proper or necessary to conduct basic analyses in situ and as fast as possible 45 miniaturisation long-term trend : A size of analytical spot from micro- to nanometre : A volume of sample and agents to submicrolitres :: microchips has analytical spots of size approx. 10 to 100 |jm :: size of human erythrocytes is 7 |jm microelectronics terminology chip (orig. thin plate of semiconductor): assembly of analytical spots on matrix (microwells or microdots containing all necessary reagencies); + other functional elements: channels, micropumps, sensors etc. array (orig. arrangement in rows and columns; system of organised elements): a way how to arrange system of analytical spots and functions on matrix microanalytical unit - whole analytical system, i.e. complete microanalyser, including dosers, valves and detectors of measured signal 46 technology of microanalytical devices =^> capture of certain target groups in sample by appropriate sensor (probe) sensor - affinity system (antibody, enzyme, protein, NA or complete biological system) detection - optical methods, electrochemical or measurements reacting on mass (e.g. acoustic waves) basic division of microanalytical devices : microreaction plates with high density of reaction spots : on-surface reactive spots : microchips and nanochips : sensors and biosensors (biochips) 47 microreaction plates with high density of reaction spots originally microtitration ELISA plate: transparent polystyrene, 8x12 (96) wells on 9x12 cm, volume of well is ca 0.2 ml contemporary microreaction plates: from 192 to 20000 microwells, volume 125 |jl to 50 nl praxis: chip devices with ca 100 analytical spots; compromise between miniaturisation and its price microdosers (based on ink-jet technology, dosing micro- to nanolitres) microdetectors (connection of microscope with photometer or fluorometer) measures signal (e.g. absorbance) in microwell of plate upside down; well is simultaneously reaction cell and also measuring cuvette measurement: method „mix and measure" microreaction plates - polymer material; casted or drilled wells by laser, laser ablation etc. microreaction plates - microchip and microarray 48 on-surface reaction spots reaction system - directly on a plate, ca 1 to 2 cm2 (not in microwell) : hundreds of microdots of size 10 - 100 pm plate material: glass, silicon, plastics (Teflon, polymethylmetacrylate, polycarbonate, polypropylene, Polyacrylamide etc.) sample loading: ink-jet technique or photolithography analytical signal measurement: reflectometry 49 microchips and nanochips microchips I_________________________I matrix/holder - ca 1.5x1.5 cm; thickness of few millimetres materials: glass, silicon, hydrophobic plastics reaction spots: wells or microdots; connected by nanochannels with valves; channels are filled with gel; : possibility to attach electrodes =^> electric voltage between well/sample and sensor sample flow-rate is monitored by sensors with laser diode (excitation by fluorescence; detection by photomultiplier; labelling of reactants/samples by fluorophores) analysis: DNA, RNA, drugs etc. fluids are mobbing on principle of electrokinesis : electroosmosis; uses el. field to move conductive aqueous solutions : electrophoresis; separation of molecules by el. field according to charge 50 external micropumps - other solution delivery possibility : but they are much bigger that microchips; difficult connection to |j-chip centrifugation analysers - solution for external pumps originally for analyses in weightlessness microchip for centrifugation analysis - LabCD material: three-layer disc, multiplicate determination of one analyte type {ca 96 same microcuvettes) content: controlling software, heating elements and microchannels, reaction wells, valves and microcuvettes liquid delivery - capillary forces, centrifugation forces mixing, dilution, washing; heating, filtering, lysation, separation of cells, photometric or fluorimetric detection modification - system of so-called surface-directed liquid flow inside microchannels - microfluid system =^> fast laminar flow, molecular diffusion channels made by photolithography LabCD function 1. sample loading 2. centrifugation preconcentration on column 3. column washing 4. column elution 5. sample in detector 52 nanochips further miniaturisation microchips =^> nanometres copying frequently function on biomacromolecules (e.g. collagen - cable, DNA - memory unit, protein membrane - pump) ultramicroanalyses (nanoanalyses) - drugs, biologically active substance, immunoanalysis, mitochondrial DNA etc. limitations - limits of analysis sensitivity and costs (rapidly increasing) complications - evaporation of sample microvolumes and agents on microchip sample: volume 1 \A, analyte 2 fmol/l = 6020 molecules of analyte volume reduction to 1 nl ^> only 6 molecules of analyte!! : mostly under limit of detection of microchip analytical method : preconcentration procedure: flow-through etc. 53 sensors use of microelectrodes on silicon sensors and biosensors e.g. resistance of thin metal layer (Au, < 30 nm) is increasing with presence of other atoms and molecules on Au surface : electrons hitting the metal film (~ mirror) are reflected : in a place where other atoms or molecules are adsorbed, electrons are not reflected, but scattered => change of Au film resistance (t); resistance = /"(concentration) use: e.g. determination of heavy metal traces in solutions (ions: Cd, Pb, Ni, Tl, Zn, their organic complexes), limit of detection in ppb (parts per billion) flow-through cells - drinking water quality, corrosive processes, but also in organisms chip sensor for penicillin silicon with pH-sensitive structure (Si3N4) D O O O O D > penicillinase --------►■ immobilisation -------*~ pH sensit, layer -------► silicon oxide ------->■ silicon penicillin + H20 penicillinase> 6-aminopenicillan acid + H+ measured with sensitive pH-layer 54 biosensors (biochips) biological system (enzyme, receptor, organ) with analytical chip use: medicine, biotechnology, check and monitoring of food and environment principle: connection of biomolecules with silicon technology EIS (capacitive electrolyte insulator semiconductor) BioFET (biologically sensitive field-effect transistors) model analytical system biosystem + analyte -^ product + electrons/protons (H+) optionally biosystem 1 + analyte -^ productl biosystem2 + productl -^ product2 + electrons/protons (H+) analysis of metabolites, personal ID (sweat) etc. 55 reference electrode 1-----1/ electrolyte kh. T membrane i ■ v----------' p *----------J ISFET ion-selective field-effect transistor '0» reference electrode analyte solution ENFET enzyme-controlled field-effect transistor 56 DNA-chips determination of target nucleic acid sequences detection of pathogens, prenatal diagnostics, forensic diagnostics, Point of Care Testing (POCT) principle: on a chip - probe with DNA sequence DNA (Watson-Crick pairing) complementary to target NA sequence only complementary sequences do interact! sample TAACGCGATTGTGTGAC probe ATTGCGCTAAGTCACTG probe is labelled e.g. luminophore => laser induction + fluorimeter fully caught red partially caught yellow not caught green ■ 4 • H 4 J 4-4 .\ t»J 41 4 ft 4 4 * J lr| a u 4 4 J J J m i i ' • ■ * *•#• J *» t > >• J 4 4 J • > t> m ii a 4 4 • >»• 4 * 1 A J «4 U * ' j • a 0 94' 4 ) ■ • • J J > -J ■ 4 4« #>#« 4 »« * (r . 4 Q d 4 a e .i * * ■i u 4 - 4 ■ •44 - :■ -J C j ujo, 04 • • * #40 J d d a * m • u -J * • ■j J * JU J oc U J t) J j 4 4 J d t ■ » 4 #4 4 1 Ů »•O« 44 > 4 0 | 4 v a • 4 u u I D • 4 O J 0 ■f • 57 immunosensors immunoassay miniaturisation (competitive, non-competitive) imunoanalysis on adsorptive support : dry chemistry method material: filtration paper, woven fleece : anchored reaction components; diffusible secondary labelled antibody sample diffuses through adsorptive material into reaction zones 1 to 5 minutes =^> coloured strips indicate test result detection: visually or single-purpose reflectometer analytical system binary single-purpose tests - yes/no analytical cassettes (cartridges) material: plastic cartridge with agents in dry or liquid state sample is mixed in a system of channels with agents detection: in place of optical cuvette the analytical signal is read : analysis of drugs, or acute analysis 58 microchips for determination of one type of analyte on microchip - set of same analytical spots - microdots microdot - immobilised primary antibody (AbJ, in surplus sensor support Abi detection Ab2 ,r^T ľff___pj W ^ sample/antigen (Ag) is added => anchored immunocomplex [Ab^-Ag] rinse; secondary labelled antibody is added (Ab2*) => => sandwich immunocomplex [Ab1b-Ag]-Ab2* detection: spectrophotometric primary antibody - antibody sensoric secondary antibody - antibody inducing multifunctional microchip - microdots with different types of antibodies => simultaneous determination of several different analytes microchips for simultaneous determination of several analytes analytical spots (microdots) - different primary antibodies (AbJ e.g. for antigen Aga primary antibody Abla, for antigen Agb primary antibody Ablb etc. rinse; mixture of monoclonal labelled secondary antibodies for determined antigens is added (*Ab2a, *Ab2b, etc.) laser microscope detection: ^■o °ľ°o °oV° ^ multianalyte microchip multianalytical immunomicrochips - POCT {ca 8 to 10 analytes), oncomarkers, allergens, hormones for endocrinology, screening of chosen analytes of blood donors in transfuse stations etc. 60 quality in clinical analysis its monitoring and management ______________________________________________________________________ „fataľ importance of clinical medicine application of norms ISO 9000 quality characteristic and desired qualities or features of product or service quality is indirectly proportional to variability of product or service : variability - diversion from design/aim examination of all steps of whole analyte determination : starting with sampling up to calculations; analyst training, agent preparation, lab-ware cleaning, waste disposition etc. concept for their proper performance is resulting, along with creation of requirements for operations with minimal diversions from prescribed procedure, including quality assurance mechanism 61 definitions according to Czech laws (CSN ISO 8402) quality total sum of properties and attributes of product (e.g. analytical kit) or of service (e.g. analysis), which guarantees ability to satisfy previously given or presumed requisites quality policy overall purpose and orientation of company activity in field of quality as defined by top management (e.g. laboratory head) quality management part of overall control function, which prescribes and defines quality concept quality system structure of organisation and responsibility, procedures, processes and sources needed for realisation of quality management; major requirement - all procedures must be unmistakable, properly documented and fulfilled; quality system is regularly monitored and complemented; all persons included should actively take part in introduction and keeping of the quality system 62 quality manual quality management in laboratory - quality manual : present in each certified or accredited laboratory comprehensive document including all aspects of laboratory activities, starting with top management down to laboratory cleaning „in-house norm" - transformation of general norms ISO 9000 and EN 45000, respectively, for good laboratory praxis (GLP), safety norms, laws and decrees etc. in accordance to particular laboratory conditions overall quality concept 63 1) laboratory and its duties - laboratory name, address, fax, e-mail, tel. numbers, name of responsible head 2) laboratory working time - way and period of sample reception 3) list of supplied services - (including analytes) 4) communication between laboratory and users - description of application forms, of result forms, rules for telephonic result forwarding, way of correction or complementation of forwarded analytical results, time period necessary for analysis procedure, including the way of monitoring its keeping 5) sampling (timing) - instructions for patient, sample transport, sample procedure up to preparation of trial sample and storage including expiration date; provision of unambiguous patient identification; way of exclusion of objectionable samples, decrees for forwarding of partial samples transfer to different laboratory sections 6) analysis procedure - written instructions for procedures used; traceability of each analysis starting with application up to laboratory results; calibration including concurrence to metrologically higher etalons 7) result interpretation - information on possible consultations with laboratory personal, assigned contact personal 8) taking part on clinical staff seminars, meetings with external medical practitioners 9) education of laboratory and clinical staff, nurses and students 10) information on procedure changes, accreditation or certification 11) taking part on research and development - concept of laboratory attendance on research and development, monitoring system and names of responsible personal 12) ways of ensuring the mandatory discretion 13) personal numbers in each category 14) information on laboratory equipment - names of instruments, its manufacturers, year of acquisition, prise, guarantee, localisation, service, service manual and its placing; calibration 15) security rules of laboratory and hospital - placing of respective decrees, records on incidents and injuries quality is not a state, but a dynamic, improving process 65 calibration, controlling and reference materials wrong analysis => wrong decision => health harming or death measuring process - respective concentration should be assigned to measured signal using calibration materials (standards) controlling-regulatory process - analysis result validation and its including into quality management; analyses monitoring in longer time scale : demands suitable controlling and reference materials 50ies - 70ies of 20th century manual analyses (photometric, titration and suchs) or semiautomatic (drainable cuvettes) : sample volume 20 - 500 |jl, agent sample 1 - 5 ml : aqueous solutions prepared of fresh analyte today automatic analysers : sample volume in microlitres, agent volume in tens of microlitres : viscosity ^> problems', calibration solutions w/o biological matrix do not fulfil :: requirements on similarity with analysed sample :: instead of aqueous solutions calibrators with proteinic matrix : certified reference materials 66 aqueous calibration solutions and standards IFCC- whenever possible - calibration using aqueous standard solutions stabilisations and protection (oxidation, bacterial contamination and so) use: enzyme attestation, verification of analytical method yield, check of photometer wave-lengths and so preparation: weighing; highly pure chemicals, redistilled water storage: glass ampoules or tight vials : under inert atmosphere of N2 or C02 auxiliary substances: albumin, polysaccharides, sugars, glycerol (enzyme stabilisation), cysteine, dithiothreitol, ascorbic acid (anti-oxidation protection), complexing agents, (e.g. EDTA, masking of heavy metals catalysing oxidation), different buffers and products of enzyme hydrolysis of substrates (increasing stability of enzymes) and conservation additives (sodium benzoate, sodium azide and such) they should not contain a considered standard as a contamination (and if, only trace and defined amount) 67 control sera and urines for operative quality management (for so-called inner quality control) liquid - frozen (- 80 °C) : originally animal sera: equine, bovine, porcine : substituted later with sera of human origin lyophilised - more stabile : prepared in at least two concentrations covering not only analyte reference interval, but also the pathologic values sera w/ attest - determining accuracy in longer time period sera w/o attest - determining repeatability parameters: pH 7 to 8, difference in content in one batch < 0.1 %, free glycerol < 0.2 %, should be sterile, residual humidity < 1 %, stability in cold 3 years, difference in content of labile components < 4 %, turbidity after reconstitution and dilution with water 1:9 measured in 1 cm cuvette as absorbance at 700 nm under 0.05 and at 340 nm under 0.2 68 serum calibrators similar composition and behaviour as analysed sample preparation: similar to control sera; addition adjusted analyte concentration lyophilisates - stability commutability with defined methods preparation and attestation: as with control sera content of individual analytes is given by definitive or reference methods which are certified by reference materials preparations for normal and pathologic analyte values multi-calibration preparations 69 certified reference materials (CRM) 70 calibration of definitive and reference methods testing/comparison of routine methods (commutability) RM - material or substance, values defined for instrument calibration, method evaluation for measurement or determination of values in materials CRM is RM documented with certificate : certified method is accompanied by uncertainty on defined confidence level analyte concentration and matrix in CRM same as in analysed sample suitability of RM for given aim is examined (ISO Guide 35) batch of CRM must be homogenous : difference between representative sample measurement must be always lower than an overall uncertainty of all measurements CRM must have stated its expiration period CRM - accompanied with attestation co-ordination by european committee within european union, e.g. institute for reference materials and measurements in Belgium (IRMM) validation and good laboratory praxis confirmation by measurement (testing) and measures of objective proof, that individual demands for given aim were fulfilled validated analytical method - gives medicinally accurate results and using analytical result within patient treatment leads not to health harming approval of overall error - sum of random and systematic errors good laboratory praxis (GLP) : internationally concerted system of assurance and control of quality : includes organisation of tests, studies and conditions, under which are nonclinical studies planned, proceeded, monitored, recorded and archived 71 operative quality management tools of QM so-called „basic seven": g§^j stem-and-leaf display - control data itemised in a way one could 3S3É quickly judge global data division H----- check list - calendar with noticed causes of faults; lines create the 1=1----- calendar, into which when and how many times the given trouble appeared is written hPareto chart - histogram with columns expressing rate of individual ■■-- fault types in descending order \ \ cause and effect diagram - graphic assay of faults and its causes flow chart, defect concentration diagram - graphic presentation qBq of instrument or process with sensitive points marked scatter diagram - correlation graph serving to estimate mutual .;.■'■ dependencies in analysed problem ■ ■*.■ i ■ control chart '/\TK regulation diagram : 1931 W.A. Shewhart : 1950 S. Levey and E.R. Jennings - clinical biochemistry simple graphic interpretation of Gaussian distribution set of data with normal distribution in interval of standard deviations -s to +s lies 68,3 % of data -2s to +2s lies 95.5 % of data {warning limit) -3s to +3s lies 99.7 % of data [regulation limit) c o 'S (0 1_ +J c a> u c o u a> >. to c (0 1p. 15 day of measurement + 3s ♦ 2$ 2s 3s x axis - time y axis - signal measured : defined portion of data must lies within the respective zones in graph : half of them must be alternatively above and below the x axis 73 cumulation only towards one side of interval =^> systematic error in analytical process : out of warning limit =^> once for a month : out of regulation limit =^> once for 18 months :: out of warning limit more frequently =^> mistakes in analytical process or process gets out of control deviations: A standard deviation (s); A deviation {B) operation of regulatory diagram average series length (ASL) 0 number of points, when inserted point indicates method out of control ASL = I//7, where p is probability, that any point crosses the control limits operative characteristic curves ß error dependence, i.e. probability of not noticing the shift ß = 0 (Ĺ - k^N) - 0{-L -HN), where 0 is Gaussian function, L is multiplicand of s according to chosen limit, k is factor respecting change of x in multiplications of s, N is number of samples 7d power function on contrary to operative characteristic curves y axis - (1-/?), so probability of denying 1.0 0J8 0.6 04 0.2 0.0 1.0 1.6 2.0 2jG 3.0 ks 1-ß N = fi^-^~~~^ /^4-~^~^ y S^ 1^^^^ ■ /jS external quality assurance (EQA) part of general quality management of each laboratory\ca once in 2 months) : method validation : laboratory monitoring its precision in comparison to other laboratories or generally valid demands what is considered? : deviations in regard to state-of-the-art, in comparison towards data gained by means of reference or definitive methods, respectively : achieved level of all committed laboratories, according to not only inter-, but also to intra-laboratory variations in recorded data : relation between data recorded and way of calibration, analytical procedure, commercial analytical kits and instrumental park used : achieved contemporary level in dependence on analyte concentration in control materials 76 international harmonised protocol for proficiency testing of (chemical) analytical laboratories : since 1992 : IUPAC, ISO and AOAC {association of official analytical chemists) IFCC material: elementals of external quality assurance precise and protocolary test organisation statistical test evaluation z-score z = (x-Xa)/s, where s is target standard deviation, x is measured quantity, Xa defined (real) quantity value and z has value of standard normal quantity z-score interpretation \z\ < 1 is good score \z\ < 2 score sufficient 2 < \z\ < 3 problematic score score Izl > 3 insufficient score 1 ' 77 importance of laboratory evaluation success of laboratory in external quality assurance its performance is inside of tolerance limits accepted by respective (inter)national authority in clinical chemistry for given time period selection Ai h collection transport preanalyt. ph. extra laboratory 20.2 % ^ IjugJ registration preanalyt. ph. in laboratory 57.1 % ('^ f1 v£y centrifugation III f distribution sample preparation preanalytical phase analytical phase postanalytical phase 78 controlled and monitored - only its own analytical procedure omitting preanalytical phase {sarnie collection, transport and storage) errors of preanalytical phase - up to 50 % X analytical procedure ~25 % the rest is assigned to so-called postanalytical phase one major error within ca 1600 analyses sample preparation (55 % caused by haemolysis), insufficient sample volume (21 %), sample confusion (12 %) and coagulated sample (5 %) major error harms life =^> more strict control of preanalytical phase Kill as few patients as possible & 56 other essays on how to be the world's best doctor, Arlan Cohn, 2004 postanalytical phase : sample and result storage : transmutation of analytical result into well-found info (raison d'etre of work) : communication with practicing medics, feed-back : meta-analysis of results analytical methods choice and optimisation___ choice of method/procedure is given by analytical chemistry development : qualitative : quantitative originally: chemical methods : number of identifiable analytes was low, only few tens since 70ies: methods biochemical/enzymatic/molecular biologic : spectrophotometry : industrial manufacturing of enzymes, analysers analytical procedure is of clinical laboratory interest : demanding requisites - analytical and also clinical needs result inaccuracy of test: including preanalytical errors, systematic errors, random errors and mistakes biological influences: intra- and inter-individual variability, errors caused by non-standard sample collection uncertainty: ability of test to give accurate conclusions (diagnosis, prognosis, or therapeutic decisions) 80 history of glucose determination: 1) redox properties in alkali media, oxidation by picric acid, ferricyanide or by Cu(I) reduction; work demanding, low sensitivity, unspecific 2) by 0-toluidine through Schiff base; sensitive and specific; o-toluidine -carcinogenic, agent contains also glacial acetic acid 3) determination using enzymatic approaches (GOD) characteristic attributes of analytical method defined and described analytical method : characteristic attributes, standardised in international norms ISO and in derived or transferred national norms 81 specific clinical requisites method accuracy in regard to biologic variability range of calibration function - minimal range of calibration function in range of analyte reference values analytical method - in principal function of given analyte in organism ecological and toxicological requisites analysis of potentially infectious biologic material never use poisons for analyses, carcinogens, corrosive or flammable materials inevitability: creatinine determination by picric acid, total protein by biuret reaction with NaOH, haemoglobin determination using haemoglobin cyanide etc. 82 analytical method attributes analytical performance characteristic : one characteristic out of sum, which are necessary for checking the precision of measuring procedure and its suitability for given aim, and which might be assigned with experimental value defined by IFCC and IUPAC accuracy or trueness : closeness of identity between average value obtained in large test set data and accepted reference value accepted reference value : value, which serves as approved reference value and which is derived as a) theoretic b) agreed (certified), based on experimental work c) assigned (certified), based on experimental collaboration d) if not a), b), c); expected value of measured quantity, i.e. median value of specified basic data set 83 bias difference between median value of test set data and accepted reference value determination1, by means of CRM or RM recovery relatively expressed difference between values measuring sample with known amount of added analyte and values of sample without addition, related to added amount precision closeness of identity between independent test results obtained under given conditions repeatability result identity determined under repeatability conditions (same laboratory, same method, same instrumentation, same operator, during short time period) 84 reproducibility identity of identification under reproducibility conditions (same method, different laboratory, different operator, different instrumentation, different time) precise specification - serves as ground for construction of regulation diagrams uncertainty of measurement parameter associated to measurement result, characterising variation of values, which might be assigned to measured quantity with good reason includes many components type A uncertainty - characterised by standard deviation derived from statistic distribution of results type B uncertainty - characterised by standard deviations derived from supposed distributions gained in experience uncertainty as standard deviation - standard uncertainty u^ resulting method uncertainty - combined standard uncertainty uc^ uncertainty - interval around measurement result (extended uncertainty U) U = k*uc, where k is extension coefficient normally distributed values and k = 2 = =^> result in given interval with probability 95 % 85 calibration set of procedures, which under specific conditions set up relation between values of measured quantities and respective calibration values (etalons) calibration function S = /"(c) analytical sensitivity - 1st derivation of the function above on concentration dS/dc = d/^/dc calculus of calibration function of calibration data by regression analysis advantage - linear dependence (limits of confidence, simpler calculations) limit of detection (Z.D, LOD) - is related to confidence limits a = ß = 0.05 ß- probability of false negative result a- probability of false positive result limit of quantification (LOQ) the lowest measurement value, for which the uncertainty could be defined; IUPAC: for LOQ uncertainty (variation coefficient) = 10 % 86 measuring interval closed value interval, which could be obtained by given measuring procedure; it is restricted by upper and lower detection limits; within photometric methods, the linear range of calibration curve is chosen 7~= /"(log c) linearity concentration range, in which analyt. signal is linear function of concentration calibration (linear function): : 10 concentrations in working interval, 3-4 measurements per concentration : test on homoskedasticity (equal distribution of their standard deviations) : regression line construction :: normal regression (homoskedasticity) :: weighted regression (heteroskedasticity) : test on linearity : calculation of confidence limits 87 analytical specificity ability of measurement procedure to measure only the desired quantity expression - as unspecificity, i.e. as effect of random sample component different from analyte causing changes in indication of measuring instrument and thus introducing systematic error interference systematic error of measurement caused by analytic interferent analytic interferent - sample component, which is also part which influences quantities; it it-self causes not changes in indication directly, but indirectly robustness (ruggedness) method ability to produce acceptable results of measurement also in a case of small deviation in measuring procedure or in sample composition 88 comparison with other methods e.g. routinely in laboratory used method and its comparison to reference method (IFCC), or with definitive method, if at disposal carry-over is not method characteristic, but says, if there is no cross-contamination within two after one running samples (mutual influence of low signal after strong and vice versa) problem of filling and flow-through cuvettes procedure: 15 analyses, 5x with water (absorbance Ax to A5) 5x with sample (absorbance A6 to A10) finally 5x with water (absorbance An to A15) A6 - A5 = A10 - A5 = A10 - An and A15 - A5 = 0, no carry-over happens carry-over - numerically in % as a ratio of carry-over 100x(A10 - A6)/(A10 - A5) 89 criteria of analytical method choice analysis in laboratory medicine : sense only in regard to evaluation of patients health two main aspects clinical usefulness - the quality demanded by medical doctors analytical determination quality - errors and uncertainties, interference and specificity of method method choice according to analytical attributes according to state-of-the-art procedures are chosen according to contemporary demanded clinical needs using analytical methods 90 according to panel experts : empirical findings of experts in specialised medical branches : compromises taking in account state-of-art more than real needs according to results of quality management : with increasing quality of analytical methods costs and laboriousness decrease choice according to clinical requisites medics experience in confrontation either state-of-art in analysis not possible to design universal requisites on quality procedures disadvantage - it differs a lot and it is not possible to find common criteria choice based on biologic variability the most relevant to clinical and analytical requisites : intra- and inter-individual variability of analytes is almost constant (even in higher age) : the geographic and temporal transfer is possible 91 choice according to clinical importance each change of health state may lead to concentration change of some analytes composition; for practical reasons it is useful to indicate only some changes - need to define useful change indication binary results test test results: positive/negative (yes/no) diagnostic importance (contingence table 2x2) : lines - results found within group sick and within group non-sick patient positive test negative test sum sick correct (cp) false (fn) N*cp + N*fn non-sick false (fp) correct (en) N*fp + N*cn sum N*cp + N*fp N*fn + N*cn N*(cp + f n + f p + en) 92 sensitivity : probability, that within sick patient a positive test result will be found sens = N*cpl {N*cp + N*fri) standard deviation ssens =V[(se/7s* (1 - sens) / N] specificity : probability, that within non-sick patient a negative test result will be found spec = N*cn / {N*fn + N*fp) standard deviation sspec = V[(s/?ec* (1 - spec) / N] non-sensitivity : probability of expecting false negative test result within sick patient : non-sensitivity is complementary to sensitivity (1 - sens) = non-sens = N*fn / (N*fn + N*cp) non-specificity : probability of expecting false positive result within non-sick patient (1 - spec) = non-spec = N*fpl (N*fp + N*cn) prediction : probability of sickness if test result is positive (or non-sickness test negative) : described by to conditional probabilities predpos = N*cpl {N*cp + N*fn) p red n eg = N*cn / (N*cn + N*fri) T -■T Z D 94 6 100 -iD 5 95 100 Z 99 101 200 sens = 94/100 = 0,94 spec = 95/100 = 0,95 non-sens = 6/100 = 0,06 non-spec = 5/100 = 0,05 predpos = 94/(94 + 5) = 0,95 predneg = 95/(95 + 6) = 0,94 94 prevalence importance : probability of sickness in defined population in defined time period prevalence- ratio of sick patients to all tested patients 100 D + 100 -.D => 200 tests => převal = 0.5 sensitivity and specificity is not changed, if number of tested changes, the prediction also changes at any time, compare only comparable testing groups T -iT Z D 470 30 500 -■D 475 9025 9500 £ 945 9055 10000 heart attack prediction D = 500, -iD = 9500 => převal = 0.05 population X cardiacs sens = 470/500 = 0,94 spec =9025/9500 = 0-95 predpos = 470/945 = 0-497 predneg = 9025/9055 = 0,997 95 incidence : sickness appearance in defined time interval (e.g. year) e.g. diabetes : prevalence in USA - 2.00 %, i.e. ca4 millions of citizens have diabetes : incidence in USA - 1.99 %, i.e. each year 398 000 of new diabetes cases efficiency : ratio of all positive results to total number of results efficiency= {N*cp + N*cn) / {N*cp + N*cn + N*fp + N*fn) likehood and likehood ratio likehood : probability is measure of phenomenon appearance within given hypothesis : likehood is phenomenon appearance measure within different hypotheses 96 likelihood quotient LQ (likelihood ratio) LQ = sens I (1 -spec) instead of prevalence and prediction value: chance (W) post - chance after conducting the test ante - chance before conducting the test Wpost = LQ * "'ante relation between probabilities and chances w= pi (i -py, p = w i (i + w) Wante =P/(1-P) = 0.05 / (1 - 0.05) = 0.0526 LQ = sens I (1 - spec) = 0.94 / (1 - 0.95) = 18.8 %ost = ĹQ*Wante = 0.0526 * 18.8 = 0.98888 %ost/ (l+^post) = 0.98888 / (1 + 0.98888) = 0.497 97 definitive methods analytical method classification based on isotope dilution (ID) and mass spectrometry (ID-MS), or on combination of ID with gas chromatography (ID-GC) : mostly not applicable into daily praxis - complex and laborious : serve mainly within attestation of calibrators and control preparations reference methods elemental, thoroughly studied and defined measuring procedure, which analytical attributes (uncertainty and bias) allow its use to check accuracy of other measuring procedures and to characterise reference material recommended methods (according to IFCC) with well described logical steps, which are ordered as they were defined and recommended by relevant authority routine methods methods, which fit none of the above groups : must be commutable with reference method commutable method - gives on representative set of native sera same results and reference method 98 analytical method optimisation optimal conditions for analysis : reaction mixture composition (type, component concentration, pH and reaction mixture temperature etc.) : individual steps and order of agents adding finding optimal reaction conditions - study of higher number of parameters, their influence is estimated there is mostly only one particular optimal combination single variable approach (SVA) relaxation method studies the reaction parameters separately; demands higher number of independent measurements; studies separately even those parameters which might correlate (may lead to incorrect conclusions) multi variable approach (MVA) studies parameters in a complex way; parameters are changed in parallel; methodically correct : demands experimental design (ED) experimental design way how to conduct experiments to get out of minimal number of points maximum of information and thus the best multivariable function description factorial design full factorial experimental design (FED) : contains all possible combinations of chosen factors parameters: number of factors and number of levels of each factor number of factors (f) is related to number of input variables (component number) number of levels(L) is number of values of each input variable {e.g. number of measured concentrations) number of points of factorial design (total number of experiments n) n = Lf 100 three-level two-factor factorial design (Z. = 3); 32 of experiments .Q .5 (0 > C ■ 4 ------« (N 0) í--------1 ----0 (0 1_ (0 C ------* —•* > variable 1 variable 1 variable 1 two-level two-factor factorial design two-level three-factor factorial design (L = 2) the simplest) 22 of experiments (L = 2) 23 of experiments fractional factorial experimental design (FrED) decreases number of experiments in contrast to FED (which is sometimes to complex and laborious) : still describes influence of each parameters and controls possible interactions : useful in cases of expensive and time-demanding experiments 101 star design : other variant of experimental design : might be FrED variant of factorial design : three-level two-factor factorial design =^> two-factor star design contains (2xf+l) of experiments, where f is number of dimensions (components) location of star design points is given by location of central point other points are located symmetrically around centre .Q .5 'Z (0 > o—9—o (N n .5 'Z 5 variable 1 •^ variable 1 two-factor star design 2xf+l of experiments three-factor star design 2xf+l of experiments 102 central and non-central composite design combination of factorial and star experimental design - complex hyper-flat central composite design - centres of both designs are equal non-central composite design - centres are not located equally n .5 'Z (0 > variable 1 five-level three-factor central composite design 2f + 2xf+l of experiments approximation methods and algorithms optimisation - intention to „discover" numerically function of output on optimised parameters - approximation black box : algorithms do not describe physico-chemical properties, but „only" numerically assign relations between variables partial least squares (PIS) PLS - MVA, values for all analysed mixture components are calculated at once canonical correlation (CC) artificial neural networks (ANN) mimicking biological system of mutually inter-connected neurons : processors - neurons : way of connection - network topology w 0) a. a c - o hidden neuron layers neurons are arranged into layers output of n-th layer are lead to each neuron in layer n + 1 first, input layer - inserts values for processing last output layer - response values of whole ANN due to change of input parameter conditions number of neurons in input and output layers are given by number of input and output variables inner, hidden layers - depends on complexity of approximated function 105 RMS Q 2 - o + i -I— connection between neurons 2 4 6 hidden neuron number represented by rational number- connection weight{w) prediction learning of output values with minimal error of values predicted by ANN comparing to experimental values - repeated setting up of numeric inputs of transformation function and monitoring of outputs into functional (real) value error - total sum of squares (TSS) sum of squares of differences of predicted and input values n TSS= Z (2j - OUT)2 i=l zx - output variable value z for given triad {x, y, z ), OUTx - its predicted (output) value, n - number of elements in training set v K J * 106 each neuron (except for output) sums values from previous layer and multiplies them by connection weight w\ NET = Z {INPX * wx ) +BIAS, i INI\ - input value, wx - respective weight value and BIASX - bias value, which is so-called bias parameter and is necessary for correct set-up of neuron value /V£7] and for whole output of network 7V£7j - neurony in neural network OUTx - transformation of NET^ (output) sum OUT. = 1 / (l + e-^J) set training/learning - n set of parameter given by experimental design testing - at least 3 sets of parameters inside of plan borders verification - at least 3 sets of parameters inside set borders (also borders them-selves) 107 analytical kits in laboratory medicine analytical kits before 1960- analytical agents prepared in clinical laboratories today- analytical agents in forms of microchips designed for special analytical instruments could not be prepared at all in laboratory requirements for analytical kit ready-for-use single-step method - one working solution two-step method - two working solutions; enzymatic assays stabile at least 12, but preferably 24 months : individual agent after opening at 2 - 8 °C stabile for a week 108 fast analysis method - signal, most commonly absorbance, until 5 min, kinetic measurement in interval of 10 s at maximum method without sample pre-treatment - deproteination, mineralisation, pre-concentration of analyte etc. analytical kit make-up manufacturing procedure - technological regulations : description of all manufacturing, checking and other operation in a form of standard operation procedure analytical kits manufacturing is organised like pharmaceuticals manufacturing liquid agents preparation: weighing (including water; more precise) vessels: glass or plastic, volume 10 to 200 ml stabile - non-corrodible, non-underflowing, gas non-permeable (oxidation, outflow of shielding inert gas) : low-pressure PE, PP, PET (polypropyleneterephtalate) etc. filling: inert gas washing, pump dosing, bacterial filters (microfiltration), labelling (bar code) 109 solid agents preparation: weighing the solid into container, solid mixture is tabletted and tablets are sealed (blister) : mills, mixing and homogenising equipment, tabletting press, granulators; under low air humidity (< 20 %) sensitive agents (enzymes, proteins) - lyophilisates : lyophilisation (freeze drying, freeze sublimation) - removal of water by sublimation in deep vacuum from frozen water solution of product; water condensates in cooler, which has much lower temperature comparing to product (higher temperature gradient) lyophilisation damper - substances allowing more easier process (proteins, cellulose derivatives etc.) photochemical PET laminate adhesive reactive zone carrier (PC) kit completion : semi-automatic, on belt electrochemical / conductive ink reactive zone carrier (PET) outer container- final label storage: at room temperature or in cold-boxes no kits for dry chemistry diagnostic strip reaction zone - on plastic strip impregnation: concentrated, liquid analytical agent soaked in absorptive material; fixation built-up plastic net absorptive support - defined properties (grammage, i.e. mass in g/m2 and absorptivity for liquids) =^> soaks any time almost the same amount of sample storage: drier (silikagel/molecular sieve) content of analytical kit name of the kit : analyte; analysis principle : storage condition, expiration period, number of analyses : information on poisons, inflammables and corrosives manual : kit principle (with chemical equations), references (lit. and patent.), agent compositions, reaction/incubation mixture composition, procedure of their preparation, storage and stability : analyte reference values, work-flow scheme, recommended calibration and control materials, notes on sample, security notes in content of analyte in sample analytical results how to express them J originally: weight, activity per volume (in biological fluid) g and mg/dl, also gram percents (g%) and milligram percents (mg%) 1977: SI system weight (g, mg) =^> molar amount (mol) concentration - mol/l (analyte with defined molecular weight) enzymes concentration of catalytic activity instead of enzyme concentration catalytic activity 1 katal - enzyme amount, which proceeds 1 mol of substrate in 1 s under defined reaction conditions concentration of catalytic activity- catalytic activity related to volume content of catalytic activity- catalytic activity related to weight international unit U (IU) - enzyme amount, which proceeds in 1 min 1 pmol of substrate under given conditions 1 U = 1 umol/min = 16.67 nmol/s = 16.67 nkat 112 enzyme/protein mass concentration : analyses in immunodiagnostics and within enzyme CK :: concentration is expressed in mg or pg of protein/I number of elements in biological fluids (cells, particles, different objects) :: numerical concentration, i.e. particle count in litre urinal sediment - arbitrary numerical concentration (arbitrary unit) : simplification of particle (elements) count expression per defined (agreed) volume after urine centrifugation, individual element counts are read in field of microscope vision in counting cell of defined volume (Biirker cell) other use: analyses of other tests in urine (e.g. protein test), in cerebrospinal liquor, in faeces (iron content) IFCC and IUPAC: abbreviation system for expression of analytical results for sample types out of SI frame 113 analysis result interpretation __________________________________________________________________ analyte - part of sample, which we determine {creatinine in serum) : determination with certain precision depending on content, stability, determination method etc. analytical method/approach - analyte determination method {Jaffé reaction with picric acid in alkali media) analysis result - value in regard to diagnosis {creatinine clearance) test on function - tested state in regard to diagnosis {glomerular filtration) index - diagnostic ratios of analyte contents analytical variability- preanalytical and also analytical phase biological variability (BV) of population - complex characteristics of variability of biochemical test results; sum of intra- and inter-individual variabilities expression: coefficient of variation in per cents (CI/) estimation: analytical approaches, which have CV < ca 1/3 BV interval 114 agreed deviation from analyte target value (TV) - instead of BV target value: average value of all result in a set after their recording : average estimated by definitive or reference method, in cases, when definitive neither reference method are not yet established, comparative method may be used relationship between analytical result and patient state healthy (normal) or he/she has analyte values deviated (pathologic) for common analytes there are so-called reference values (formerly normal or physiologic value) í - -i p : reference values of reference individual - donor s r l ľ : reference groups 10M I 111 h ml I íTrh : reference population 0.2 0.8 1.4 0.2 0.8 1.4 ALP activity acquiring: sample analyses of persons, who are classified as healthy reference (physiologic) interval - laboratory test values, between which lie majority of the values obtained by reference population measurements 115 analytic set functional sets of individual analytical methods clinico-biochemic queries of medics : liver set, set for fat metabolism, set for glucose tolerance disruption etc.) other reasons - organisation, security, economy acute (statím) analysis determination of one or group ofanalytes as soon as possible and non-stop glucose (diabetes), creatinine or urea (kidneys), bilirubin and aminotransferase ALT (liver tests), creatine kinase and troponin (heart), a-amylase or lipase (pancreas), acido-basic blood equilibrium, basic toxicological tests and basic urine tests basic biochemical test basic (screening) tests on analytes characterising function of main body organs total protein, bilirubin, creatinine, urea, glucose, cholesterol, uric acid, ALP, aminotransferases ALT and AST, event. GMT or CK 116 basic haematoloaic tests determination of blood picture haemoglobin, erythrocytes, haematocrit, leukocytes, thrombocytes, reticulocytes, osmotic resistance of erythrocytes, basophilic granulation of erythrocytes etc. organisation division of analyses into sets immunochemical tests Immunoelectrophoresis serum proteins, al-foetoprotein, immunoglobulins A, G, M, prealbumin, al-antitrypsin, a2-macroglobulin, transferrin, caeruloplasmin, CRP, prostatic specific antigen etc. radioimmunoanalvtical tests thyroxine, triiodthyronine, thyrotropin, luteinisation hormone, follicle stimulating hormone, prolactine, choriogonadotropin, estradiol, progesteron etc. test of cerebrospinal liquor chemical determination ofanalytes + different special tests 117 basic urine test pH, nitrites, proteins, glucose, bilirubin, urobilinogen, keto-substances, osmolality, erythrocytes and haemoglobin, leukocytes, urine sedimentation supplementary analyses morphologic analysis of urine sediment (epithelial cells; granular, wax, cellular, erythrocytal, leukocytal, bile casts; pseudo-casts, spermatids and microbes (yeasts, bacteria, trichomonades, mould) drug monitoring monitoring of those pharmaceutical levels, which in higher doses are toxic or over-dose is meaning, or because patients may have lower tolerance to them special analysers the most monitored pharmaceuticals - digoxin, Phenytoin, phenobarbital, valproic acid, diazepam, theophylline, gentamicin, tobramicin, methotrexat, Ciclosporin etc. drugs of abuse instrumental method (HPLC, GC, MS, CZE etc.) alcohol, amphetamine, barbiturates, benzodiazepines, cannabinoids, cocaine, methadone, opiates, antidepressives, anabolic steroids etc. selected instrumental analytical methods in laboratory medicine spectrometric methods : UV-Vis, fluorimetry : AAS, AES : mass spectrometry electrochemical : coulometry, potentiometry separation methods : chromatography (liquid, gas) : electrophoresis (gel, capillary) 119 high performance liquid chromatography (HPLC) _____________________________________________________________________________________________________________ principle separation based on different distribution constants of analytes in two-phase system (solid-liquid) distribution ratio D D = Cs /CM C^ - analyte concentration on solid (stat.) phase, CM - in liquid (mobile) phase retention time tR : time, which it takes to analyte to go through column void (dead) time tM : time, which it takes to mobile phase itself to go through column, or non-interacting substance (its tR = tM) sorption - analyte from mobile phase is adsorbed on stationary phase desorption - reverse process chromatography - dynamic equilibrium of analyte sorption and desorption four main sorption mechanisms: adsorption, distribution (extraction-like), ion exchange on ion-exchanger and steric exclusion (stationary phase with defined porosity, separates substance according to their size and molecular mass) 120 adsorption - surface effect caused by electrostatic interactions (weak bods; H-bridges, bond dipole-dipole and bond dipole-induced dipóle) analytes compete with mobile phase fox limited number of binding sites on adsorbent surface substance chromatographic separation - symmetric or Gaussian concentration profile in direction of mobile phase motion, i.e. zones or peaks quality of chromatographic separation : column efficiency number of theoretic plates N N- 5.54*(ŕR / l/l/)2 W- peak half-width height equivalent of theoretic plate H H- L IN L - column length high H =^> efficient s. of multi-component substance mixtures | laminar flow of liquid c "55 resolution Rs Rs = 2*ArR / (W1 + W2) | : separation of two neighbouring peaks Rs > 1.5 satisfying, Rs = 1.5 is so-called baseline separation time 121 stationary phases f SF) chemically modified silikagel or different copolymers silikagel Si-OH - polar and weak acid : modification by alkylchlorosilanes with alifatic carbon chain length 1 to 18; hydrolytically stable siloxanes with typical bonds Si-O-Si-C : most frequently used phase - octadecylsiloxane (ODS or C18); so-called reversed phase mobile phases f MF) ace. to polarity: /7-hexane, cyclohexane, methylbenzene, chlorinated carbohydrates, tetrahydrofuran, acetone, acetonitrile, /so-propanol, ethanol, methanol, water... individually or their miscible mixtures MF flows under pressure up to 40 MPa, flow rates 0.05 - 2.00 ml/min reversedHPLC- methanol or acetonitrile mixture with water or buffer solution pH of aqueous part!! - ionised substance forms have lower affinity to C18 122 HPLC columns conventional- stainless steel, length 3 to 25 cm, i.d. 3-5 mm microcolumns- length 5-50 cm, i.d. 0.5 - 2 mm capillary HPLC signal detection : in MF after elution from column photometers UV-Vis (PDA/DAD), fluorimeters, refractometers, electrochemical detectors, novel mass spectrometry HPLC detector sensitivity : refractometric and conductivity detectors 5.10-7 g/cm3 : photometric detectors 10~10 g/cm3 : fluorimetric and amperometric detectors up to 10~12 g/cm3 123 HPLC application in laboratory diagnostics fragile chromosome X syndrome cause of inherited mental retardation (2nd most often after Down syndrome) manifestation: facial dysmorphy - big ears, big face w/ accent, chin and jaw dystropies: stereotype, ill communication, hyperactivity and bad spatial orientation defect gene FMR1 on chromosome X (fragile X mental retardation gene 1) mutation: trinucleotide repetition multiplication (SNP, single nucleotide polymorphism) : Fra X - CGG repetition multiplication and following cytosine methylation : Cyt methylation of CGG sequence =^> gene expression suppression =^> Fra X according to content of deoxycytidine monophosphate (dCMP) and its methylated derivative (mdCMP) and according to number of CGG repetitions, gene FMR 1 might have three stadia: FRM 1 state dCMP [%] mdCMP [%] CGG number normal 70 - 100 0-30 6-53 premutation 50-70 30-50 < 200 mutation 10-50 50-90 > 200 estimation of dCMP/mdCMP content ratio diagnostics of Fra X syndrome Cgi|| ^> QNA endonuclease Mso I^ nucleotide triphosphates exonucieasewiy nucleotide monophosphates (mdCMP / dCMP)! vs. (mdCMP / dCMP)2 NH2 dAMP 1 N \----i-OH dTMP HhT "^-CH. 0=L OH dGMP ). HM HjPOj-O-CHj----( mdCMP has R = CH, dCMP o HjN-----(' N OH -CH3-0-P0JH= HO- %mdCMP = mdCMP / (mdCMP + dCMP) checking ratios (mdCMP + dCMP) / dGMP = 1 dAMP / dTMP = 1 improving sensitivity in order of magnitude UV-Vis =^> fluorescence detection derivatisation by dansyl chloride < 70 ! 60- 50-40 30 20 10-0- dC -■---------------1---------------■" 5 10 ->---------------------------------------1---------------------------------------■" 15 20 tR (min) 125 capillary electrophoresis (CE) principle separation according to different electrophoretic mobilities of analytes in potential gradient of electric field d - distance, along which analyte migrates after introduction of potential E between two electrodes with distance S in time t d = M*t*(E/S) p - electrophoretic mobility (it is function of charge, molecular mass and shape; friction forces - slow-down migration in viscose electrolyte) separation Ad = (px - |J2) * t * (E / S) the most utilised methodics CZE - capillary zone electrophoresis PAGE - Polyacrylamide gel electrophoresis 126 CZE CZE - capillary zone electrophoresis : silica capillary of small diameter (50 - 75 |jm) __________ : negatively charged surface {silanolgroups) (+} (+) (+} : voltage ~ 10 - 60 kV &&&& compensated by buffer cations =^> Stern electric double-layer potential gradient =^> hydrated cations are in motion =^> shift of a whole electrolyte (EOF) =^> carries towards cathode all present substances (neutral and also ionic) >k >k i '----------------"-E0F EOF rate -Tw/T buffer pH and voltage; 4> w/ viscosity °Ji_ ■+- EOF+N background electrolyte (conductive medium) - buffer .^Vä1---------->-eof+c+ (BE) ■----------*■ E0F-A" : fundamental influence on constant mobility of separated substances H3N+-CH2-COOH (H2N-CH2-COOH H3N+-CH2-COO-) H2N-CH2-COO" 127 Separation of neutral species - adding tenside into BE micelle creation =^> encapsulating neutrals =^> charged micelles are separated separation based no their different solubility in micelles or based on differences in distribution coefficients of analytes between aqueous and micellar phase signal detection : UV-Vis - sensitivity 10"13 up to 10"16 mol (10~5 up to 10~8 mol/l) : amperometric detection - 10-11 mol/l : laser induced fluorescence (LIF) - 10~14 up to 10~16 mol/l :: derivatisation by fluorophore 128 PAGE - Polyacrylamide gel electrophoresis migration in polymeric matrix (agar, starch, agarose, polyavrylamide) : separation of macromolecules modes : isocratic, gradient : denaturing (SDS; Lämmli) : non-denaturing ey °k protel^v - Oy SDS 1 -> 3 ■ !A -,.—2—, | 23.13 kb 9.42 kb 6.56 kb 4.36 kb 2.32 kb 2.03 kb proteins detection: Coomassie Blue, silver, SYPRO ruby, Cu(II), Zn(II) staining, densitometry NA SYBR green, ethidium bromide, Acridine orange 129 CZE application in laboratory diagnostics metabolic disorders : disrupted metabolic pathways (blocked, absence of enzyme /genetic/) :: presence of products of defective metabolism in urine and blood determination of purines and pyrimidines content diagnostics of xanthinuria and hypoxanthine phosphoribosyltransferase deficit BE - borate buffer 30 mmol/1 pH 10.2; hydrodynamic injection 10 s, separation voltage 15 kV, temperature 25 °C, UV-detection at 260 nm 0 N N xanthine o hypoxanthine N V N ^N' U C (D .Q i_ O .Q (D O N N- 0 'N A isr^o unc acid r i C c + (D X o - 2 "Š Q) C C (D "G (D Q. >« .£ C !E ■M 1 - S 3 Ol C (D ■g 3 X 3 ..._________yi ^---------- ' 1 1 1 migration time [min] 130 mass spectrometry (MS) : physical method; determination of molecular or atomic mass of analyte principle molecules of analyte are ionised in gaseous phase; then they are separated either in time or space according to mass to charge ratio {m/z) ionisation = mass analysis = ion detection ionisation - by field or high energetic particles (electron, photon, atom) soft i/5. hard ionisation mass analysis ion source ion optics magnet magnetic sector time-of-flight detector laser ion. chamber t ion extractor Hr flight tube reflectron ion detector detector 4^ ion detection - system of dynodes 131 tandem MS serially connected several mass analysers + secondary Ionisation- collision cell : first analyser separates ions according to m/z : ion selection at certain m/z : secondary ionisation-fragmentation : fragments separation in second analyser : fragment ion detection Val Lys Val Leu Gly Asp Val He Olu Val His Gly Lys _^ h2 b3 b4 b5 b6 b7 b8 bS bl0 b11 bl2 ti. Ü 0 l t l Hjc-c-w-c-c ^12 yi1 yi0 y9 y8 y7 h y5 \ h \ yi analysis of proteins and nucleic acids MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight) H H 0 í i I -N-C-C I H H 0 I I I H-C-I H L .N II M 0 N-C-C I H H 0 i I J Íi-C-C I R» H H 0 i I i tí-C-C H H O 1 i ■ N-C-C H H 0 I i i N-C-C i H H 0 l l l N-C-C-OH R., MALDI PSD TOF post-source decay 80 -, sP 60 -O* c hormone identification £ by tandem MS b b, [M+H]+ a. _LJL-L 4 : y3 a. ml a. iMii^^jyiu^i^ y7 ^ j b y a wAw^^kJwÍvW* uU* W**M*~,*)4i U 200 400 600 800 1000 , 1200 m/z an, bn n = 1 pGlu-His- 8 -Trp- -Ser- -Tyr- ■Gly- ■Leu- -Arg- ■Pro- -Gly-NH; 8 1 =n yn luteinising hormone-releasing hormone (LHRH) 133 -----------------------:--------------------1 VI immunochemical analysis in praxis of clinical diagnostics I immunity system (IS) - information system : responsible for identity and integrity of individual components: lymphocytes and antibodies (Ab) localisation: in blood and lymphatic circulation function: tolerate intraneous structures, label and remove extraneous : recognition of intraneous from extraneous : removal of extraneous: micro-organisms, cells, tissues, proteins and substances with antigenic activity antigen + antibody interaction : analytical use of immunity system - affinity (specific) interaction : non-covalent : reversible : variable 134 antigen {antibodygenerator) (Ag) : contains determinants (epitopes) =^> induces imm. reaction (immunoqen) :: macromolecule (> 8 kDa) :: macromolecular carrier + hapten bacterial toxins, lipopolysaccharides, agglutinins, rheumatoid factor, endotoxins, allergens antibody (Ab) : recognise determinants =^> response of IS to Aq : specific Ab reacts with particular Ag - labels it : binds to determinants by means of binding site (paratope) :: immunisation - exposing IS to new Ag =^> creation of new Ab monoclonal, polyclonal Ab - equivalence of epitopes Ab cross reactions - interaction of one paratope with similar epitopes 1: immunoglobulins IgA (15 - 20%) - on the surface of exocrine gland mucous membrane Ig D - activation of B-cells IgE - w/ allergies; double binding: allergen-IgE-glycoprotein IgG (75%) IgM (3 - 10%) - intravascular space antibody - detail of structure antigen bound to Fab fragment antigen I light (L) chain I heavy (H) binding site A. saccharide disulphide bridges j Fc fragment 136 interactions : direct - precipitation, light scattering : indirect - agglutination, complement bond : labelling - immunoassay :: homogeneous :: heterogeneous : direct : competitive : indirect, sandwich immunoanalysis medium detection within direct and indirect immunoanalyses : visual : optical (nephelometry, turbidimetry) : mass spectrometric : solutions : gel (immunodiffusion) : immobilisations & their combinations :: tissues within labelling immunoanalyses : spectrophotometric, fluorimetric : radiometric : electrochemical 137 direct immunoanalvsis - precipitation reactions bivalent Ab (precipitin), macromolecular Ag (precipitinogen), 1:1 precipitate - visible coagulate originated in Ab and Ag molecules only Ag .-. 2.0 Ol < U) CD 10 ? o,s y < U) CO fcfl / / s v B desegregation substances - prevent precipitation 0,0 02 0.4 0 6 0.8 BSA [mg] precipitation curve direct immunoanalvsis - agglutination reactions corpuskular Ag -----------M polyvalent Ab (agglutinin) corpuscular Ag (agglutinogen) agglutinate - visible coagulate originated in macroscopic particles corpusculation Ag: immobilisation on corpuscule (macroscopic particle) : haemagglutionation, cytoagglutination, latex; complement system 138 qualitative methods ring p. - precipitation ring on inter-phase Ag - Ab plate p. - „plate" immunity reaction u V u "O I' U) 3 it paraffin Ag solution precipitate Ab agarose Oudine method - two gel layers, with Ab or Ag Oakley-Fulthorp method - two gels, with Ab or Ag, detects more [AbAg] plasma Ouchterlony method : double radial immunodiffusion :: concentric system of pits Ab in the middle Ag around Ag . 1:64 Ab A9 Ag . 1:2 . 1:32 V^L^/ A9 1:4 • ft Ag 1:16 Ag 1:8 non-identical album partially identical anti-albumin anti-fibrinogen DNP-Ibumin nti-DNP + anti-albumin IgGK identical anti-Y + anti-K Williams & Grabar method : Immunoelectrophoresis anode gel w/ Ab 4-** precipitate cathode start :: starting pits with Ag, lengthwise pit with free diffusing Ab counter-directive immunoelectrophoresis : on opposite ends Ab and Ag, precipitation zones 140 haemaqqiutination inhibition test (HIT) S3"1?'6 ..- U T ^ t ,| anti Hu-IgG k ». + ä Gl epitope + A X Hu-IgG Gl epitope sample ^ Hu-IgG "^ ^ other epitope no agglutination positive - blood sediments Ab presence =^> no agglutination negative - agglutinated erythrocytes Ab absence =^> agglutination indicators or j^Hu-IgG eryth. Rh+ eryth. with Hu-IgG sample * ^* A anti Hu-IgG child direct CT mother or Coombs test (CT) : revelation of antibodies against red-blood cells : determination of Agx by un-complete Ab' : primary complex: Ag^b' : secondary complex: Ag1Ab'Ag2 indirect CT step 1 Coombs reagent oj + A sample + V^ %5J O step 2 Vtf* ^ 141 quantitative methods gravimetric p. - p. weighting, nephelometry, turbidimetry Mancini method : radial immunodiffusion :: gel contains Ab :: line of pits ::: standards (calibration) + sample Laurel method : rocket, continuative, cross IELFO; electroimmunodiffusion :: gel contains Ab, starting pits with Ag; flame shape - semi-quantity Clarke & Freeman method - 2D IELFO : ID separates Ag, 2D interactions with Ab in gel in 90° tandem IELFO : similar to 2D IELFO with standard amounts of Ag ___ ©^ ---- o-— tl f ; ad in gei Ag 1.0 mg 2.5 mg 10.0 mg if Ag sample 15 mg/ml diameter [mm2] A (+) 1. dimension (-) á (+) start * ■ ..... * II II I o gel w/AB \ j y n 142 immunoanalvsis with labelled reagents immunoassay; serum neutralisation univalent Ab, soluble Ag : suitable Ab - monoclonal : suitable label (Ab, Ag) - fluoro- or chromophore, radioactive isotope, enzyme : separation system - immobilisation : detection : standard or control measurement 143 simple immunoassay : immobilisation of Ag : known amount of Ab : bound of Ab to Ag : Ag contant determination :: directly - labelled Abt :: indirectly - labelled Ab2 against No1 r •____• Ag // ?? concentration i labelled Ab O substrate Ag + Ab* ^ [AgAb*] advantages: low Ab consumption disadvantages: sample immobilisation, labelled Ab, low specifity, low selectivity (only within direct detection) 144 competitive immunoassay limited reagent assay : limited number of binding sites on plate with Ab : unknown amount of Ag : binding site competition with known amount of labelled Ag :: determination of labelled Ag either free or bound => unknown amount of Ag m<% yÍ^y ffÝf IffÝf concentration • Ag Y Ab ^labelled Ab O substrate 2Ab + Ag + Ag* ^ [AbAg] + [AbAg*] advantages: low Ab consumption disadvantages: low specificity, low selectivity 145 sandwich immunoassay, immunometry reagent surplus assay : two or more Ab guarantee IA specificity : theoretical limit of detection is one analyte molecule :: unbound labelled Ab is used to quantify the analyte YYYY • Ag Y Ab X labelled Ab O substrate x Abi + y Ag -> y [AbtAg] + (x-y) Abt z Ab2* + y [AbiAg] -> y [AbiAg]-Ab2* + (z-y) Ab2* advantages: elevated sensitivity, elevated specificity disadvantages: labelled Ab, Ab wasting YtYÝ (D C Ol "55 concentration FIA - fluorescent immunoassay fluophore labelled Ab or Ag design homogeneous : A fluorescence properties of labelled Ab/Ag when [AbAg] is created heterogeneous : fluorescence of bound labelled Ab/Ag after washing-out the unbound one fluophores used: fluorescein, fluorescein isothiocyanate (FITC), rhodamine, umbelliferon, 8-anilin-l-naphthalen sulphate (ANS), lanthanoid chelates (Eu, Tb, Sm) advantages disadvantages : specificity : sensitivity : probe size background (quenching) instrumentally demanding limited choice of labels 147 chemiluminiscence acridinium esters - need no catalysis CO-0-3 -ROH CO-0 CH. CH. -CC, I +tn CH, luminol, isoluminol - needs hydrogen peroxide; peroxidase R1 O R Pi NH NH POD, H2Q^ R pH (10-11; co-o' CO-O' + N2 + hv + H20 O enzymatically induced luminiscence component: 1,2-dioxoethane 0-0 y CH» til I 0PO(OH^ ♦V -H^O, ď&-&-\* +■ hv 148 5 electrochemoluminiscence NHS ester of Ru(bpy)32+ anchored on magnetic carrier label oxidation on Pt or Au electrode at 2 V + TPA (tripropylamine) in buffer - regeneration bioluminiscence) luciferin/luciferase HO S s N ^1 ATP, Mg(II), Q2 HO "n-^COOH luciferase * N N I i 0-0 HO -CO, advantages : S/N ratio : sensitive : probe size disadvantages : instrumetally demanding 149 RIA - radioiosotopic immunoassay radioisotope (unstable / radioctive isotope) labelled Ab or Ag design: : heterogeneous, conmpetitive isotopes used: 125I - protein Ag; 60 days; 14C - haptens; 3H - steroid hormones other isotopes - 75Se, 57Co detection: according to radiation - a and ß {scintillator) or y {counter) advantages disadvantages : flexibility : toxicity : sensitivity : shelf life : probe size : waste disposal EIA - enzyme immunoassay enzyme labelled Ab or Ag design homogeneous : competitive Ab + Ag-E + Ag -» [Ab*Ag-E] + Ag-E + Ag heterogeneous : competitive : direct : indirect/sandwich enzymes used: alkalic phosphatase, peroxidase, galactosidase, glucose oxidase, dehydrogenase, lysozyme and ma late dehydrogenase advantages disadvantages : versatility : unstable : purposeful : probe size : signal amplification : heterogeneous 151 immunodiagnostics _____________________________________ immunoanalysers : fully automated immunoanalyses : mostly based on heterogeneous sandwich immunoassay (EIA, FIA) :: every manufacturer has its own design modifications (patents) : relatively low costs (mass Ab production) 152 immunostrips : specialised one-target disposable immunoanalyses : mostly based on heterogeneous sandwich immunoassay (EIA) : relatively low costs (mass Ab production) sample flow sample plug for reader plasma separation membrane myla support substrate electrode immunoreaction zone wx AÍ1II point-of-care meters based on immunoanalysis : specialised single- or multi-purpose mini-immunoanalysers : mostly based on heterogeneous sandwich immunoassay (EIA) : originated in usage of immunostrips 154 immunochips further miniaturisation classical advantages of chip technology secondary Ab waste buffer grid with immobilised Ag OptoLabCard+Skinpatch :: lab-on-foil > The SmartBioPhone™ 155 immunohistochemistry immunocytochemistry application of immunologic methods to study tissues and isolated cells : tumour or pathogenic changes detection biopsy + Z -k N2(/) freezing W&r- i thickness 4-8 |jm cryostatic slicing mostly flurophore, metal or enzyme labelling : direct, indirect, indirect three-stage (Ab-„bridge") - signal amplification + chromogenic substrate Ab,* ALPase + chromogenic substrate, Ab: bridge BSPOX method B - biotin, S - streptavidin, POX - peroxidase APAAP method 156 application of immunologic methods to study IS cells : detection of pathogenic changes in IS mostly Ab flurophore labelling : flow-cytometry cellular suspense side photomultiplier colour filters scatteringŕ9ŤFj _ photosensit. GHWHs forward scattering!^ cell separation control fluorescence intensity antiCD4-Ab* 01 c 'Z (0 u (A ■o "35 granulocytes m°n°cy*es --- lymphocytes forward scattering 157 protein analysis in clinical biochemistry tertiary structure quaternary structure VII, primary structure protein structure secondary structure . primary structure - amino acid sequence secondary structure - spontaneous assembly tertiary structure - 3D (induced by chaperonines) quaternary structure - supracomplexes 158 specific - determination of particular protein non-specific - determination of total protein or just one protein non-specific determinations Kjeldahl method polypeptide => ammonia, p. content is defined as organic nitrogen peptide^ ^ tJtv • ammonia collection vessel sample is mineralised by concentrated sulphuric acid in presence of catalyst; aminic nitrogen =^> ammonium ions ammonium ions are turned into ammonia by heating and are collected by distillation in collection vessel, where it is again turned into ammonium ions ammonium ions are determined by neutralisation titration 159 spectrophotometric determination (SPEFO) 205; 260 and 280 nm : peptide bond; aromatic structures absorbance is influenced by : secondary, tertiary and quaternary structures : factors such as pH, ionic strength etc. (un)known sample : calibration on BSA =^> absorption coefficient at 205 or 280 nm unknown sample with possible DNA contamination : measurement at 260 and 280 nm : concentration (mg/ml) = (1.55 x A280) - (0.76 x A260) non-invasive (consumes no sample) : imprecise, easy false positive results - contamination SPEFO - biuret method : invasive, time consuming, high sample consumption biuret agent: 25 g potassium sodium tartrate 0.75 g copper sulphate 5H20 1.25 g potassium iodide :: all in 100 ml 0.2 M NaOH : sample of 1 - 10 mg/ml protein : calibration on BSA : add biuret agent, stir and let stay for 20 min : absorbance at 550 nm o c \ tartrate - solubility of Cu(II) in alk. # iodide - catalysis Cu(II)^>Cu(I) reduction ? lipaemic serum - false positive results also these groups react -CONH2, -C(NH)(NH2)- and -CSNH2 161 o-r; / \ NH2 N-h I H NH? Nilv II biuret agent glycine AT ■m- _ 2- 0 <: o=c \ / \ Nil;, NM NM;, 0=C I UN Ctl y \ UN Nn c=o Nil o=c ^N^ c=o ?Nft egg white _ 2- ü C :h—áíí) H—CO- —CO— N N —CH— @- CH C=0 2 Na SPEFO - Lowry method : variation on biuret method in alkali media with copper ions : after adding Folin-Ciocälteu reagent, it binds to protein, then is slowly reduced to heteropolymolybdenite blue by means of Cu(II)-catalysed oxidation of aromatic acids; colour changes yellow to blue : 100 pi sample + 1.0 ml Lowry reagent (alkali CuS04) : incubation 30 min at 25 °C : + 100 ml of Folin-Ciocälteu reagent : incubation 30 min at 25 °C Folin-Ciocälteu : absorbance at 595 nm 750 m! water; 10Q g Na2W04; 25 g Na2MoO4;50 ml 85% phosphoric acid; 100 ml cone. HCl; 150 g Li2S04 + few drops of Br2 proteins are different, calibration on BSA is not sufficient interferents: barbiturate, CAPS, CsCI2, citrate, cysteine, diethanolamine, dithiothreitol, EDTA, EGTA, HEPES, mercaptoethanol, Nonidet P-40, phenol, polyvinylpyrrolidon, sodium deoxycholate, sodium salicylate, thimerosol, Tricin, TRIS and Triton X-100 162 SPEFO - modified/Smith-Lowry method : similar procedure as for Lowry method : absorbance at 562 nm : sensitivity not higher, but lowered influence of contaminants method very sensitive because of precise pipetting mechanism o o n m C-ONa C-ONa bicinchoninic acid - BCA 1. protein + Cu2+- OH- ■*-Cu1+ BCA + Cu COO- COO- BCA-Cu1+ complex 163 SPEFO - Bradford method A absorbance of Coomassie Brilliant Blue G-250 after binding to protein : calibration on BSA (1 mg/ml) : absorbance at 595 nm without incubation not sensitive to interferents, except for high cone, of tensides HN significant difference in protein-to-protein absorption suitable calibration! H^N^Y'V*03" other modifications of SPEFO method colloid gold *>^<& ^w\4 L V 69S4a Í 8^6 47 | ° L.1098.74 200 300 400 500 S00 700 800 900 1000 1100 1200 Edman sequencing separation of phenylthiohydantoine derivatives : ion-pairing HPLC : RP-HPLC : CZE other methods of identification - melting point comparison Edman vs MS MS advantages - low sample consumption pre-separation on PAGE ES advantages - automatable routine method 167 / \_N-C-S phenyl isothiocya nate + O O I I HjN-CH — C — NH— CH — C — N-terminus of peptide __ H i N-N-C-S O O \=/ I I i H-N— CH — C— NH-CH-C- Fti H, phenylthiocarbamoyl derivative of peptide chain EH* 1 H O a I I N —c—s + H3rt— CH— c — H- A r, peptide chain shorter of one unit O N—C —S / \ C NH 1 CH phenylthiohydantoine derivative of R± B gel electrophoresis (PAGE - Polyacrylamide gel elfo) : separation in gel + suitable staining denaturing PAGE - SDS normalises charge, separation according to Mw :: densitometry staining types : silver staining- not quantitative : Coomassie Brilliant Blue- semi-quantitative : SYPRO ruby- quantitative mass spectrometry : quantitative using isotopically labelled internal standard capillary electrophoresis H PLC : UVdetection- unsuitable : fluorescence detection - precolumn derivatisation: dansyl chloride, o-phthaldialdehyde 168 enzyme analysis holoenzyme forms of one enzyme - isoenzymes enzyme ~ biocatalyst coenzyme (cofactor) + apoenzyme E + S <—> E-S* <--► ES <--► E-X* <--► EP <--► E-P* <--► E + P >« Ol L. « c E-X* E + S EP E + P reaction coordinates active site O-!?? f* enzyme ö-o-ä substrate (S) binds into active site =^> complex enzyme-substrate {ES} {ES} =^> P; enzyme regenerates bond ES - highly specific; depends on AA composition and cofactors enzyme effect - catalytic activity; concentration of catalytic activity 169 enzymatic reaction inhibition allosteric enzymes uncompetitive competitive inhibitors : specific and unspecific : reversible and irreversible activated enzymatic reactions & 9? $5 - ' inhibitor *_j acorn petitive : cations: Ca(II), Mg(II), Zn(II)... : anions: CI(I) : organic substances: metabolic intermediates and hormones 170 mechanism of enzymatic catalysis lock and key . general acido-basic catalysis : nucleophilic and electrophilic catalysis active site substrate €V^i-€*l enzyme ES complex enzyme product kinetics of enzymatic reaction w/ one S substrate induced fit W-SB- w \ V V enzyme ES complex enzyme product E + S * k-, Kí (-►EP)-> E + P = - d[S] / dt = k1*[E]*[S] - k_!*[ES] = d[P] / dt = k2*[ES] y= Kn«*[s] / (/fm + [s]) Michaelis-Menten equation Vmayi - maximum rate Km - Michaelis constant; concentration of S, where v = V2 Vr max 171 enzyme + substrate short period for ES establishment, so called lag-phase (induction period) [E] of enzyme decreases, [ES] increases up to steady state (ms) =^> [ES] = const, [P] = 0 o '-4—> u o u [S] [P] [ES] time [s] C o ■4—> ru s_ ■4—> \ lag-phase [P] ^ (D U \ [ES] C \ o f u S i [E] i y 1 s • __y time [ms] enzyme kinetics zoom temperature reaction conditions influence range of temperature optima: 0 - 150 °C :: common range of temperature stability: 20 - 40 °C to increase temperature stability :: carrier immobilisation; :: biotechnological / genetic engineering enzymatic reaction rate - Arrhenius law EÜ temperature [°C] given by number and quality of (pK) dissociable groups : pH optimum of enzyme with synthetic substrates in vitro :: is different from pH optimum for enzyme in vivo 173 : often important for not only pH optimum set-up :: synergic effect of buffer - effect cummulation : simple buffers buffer as second enzyme substrate " amPno|y1:es catalytic concentration determination for alkalic phosphatase (ALP) enzyme activity fundamentally depends on buffer type : carbonate - ALP works as hydrolase : amino alcohol - ALP works as phosphate transferase amino alcohol buffer is simultaneously the second enzyme substrate buffer catalytic concentration determination for y-g/utamyl transferase (GMT) amino acid is the second enzyme substrate as glutamyl group acceptor : without amino acid - GMT works only as hydrolase : optimal second substrate - glycyl-glycine : simultaneously buffer + synergic influence on GMT; pK ~ opt. pH for GMT buffer as specific moderator with enzyme determination turbidimetric enzymatic determination of fibrinogen using proteolytic enzyme batroxobine :: reaction rate specifically increased in presence of glycyl-glycine in ca 80 % :: enzymatic reaction has pHopt = 8, ~ pK^ glycyl-glycine =^> simultaneously buffer and activator, synergic effect buffer as unspecific reaction moderator enzymatic determination of glucose using glucose oxidase (GOD), peroxidase and oxidative copulation :: TRIS: reaction id slow and slow is also reaching of the equilibrium :: phosphate buffer: reaction rate is dramatically increased and equilibrium is reached quickly 175 buffer as enzyme activity stabiliser urea determination by urease :: -SH group in active site =^> buffers combining EDTA (acidic buffer component) with different bases; EDTA shields enzyme before inactivation by heavy metal traces buffer as problem source in enzyme analysis determinations using ALP :: diethanolamine and aminomethyl propanol: deterrent buffer examples ::: honey-like substances, unable to crystallise and difficult to purify ::: contain impurities interfering ALP determinations, which is metalloprotein; complexation of Zn(II) cations in active site of ALP 176 moderator activators or inhibitors inhibitor as an analyte: degree of inhibition points to inhibitor concentration substrate practical reasons =^> synthetic substrates : structurally defined, industrial manufacturing : substrate with auto-indication properties 4-nitrophenyl phosphate ^=^> 4-nitrophenol : substrate =^> product, which is substrate of indicator reaction 1-naphtyl phosphate ^> 1-naphtyl + 4-aminoantipyrin => quinone monoimine 177 determination of Km and Vmax enzymes as analytes linearised Michaelis-Menten equation according to Lineweaver and Burk 1/^=1/ Knax + Km /as\ alkalic phosphatase (ALP) absorbance at 400 n m AA/min tg a = KM / V max 1/[S] incubation mixture : ALP + substrate 4-nitrophenyl phosphate : alkali medium : buffer N-methyl-D-glucamine, pH 10.1 at 37 °C incubation mixture contains always the same amount of enzyme and buffer, it is started by substrate, which concentration is gradually increased : AA measured between 30 and 120 s after start Km and Vmax estimation may be done in graph of linearised equation 178 enzymes as analytical agents enzyme : fast, elegant, highly specific chemical reaction under mild conditions CD U .o o to .o uricase : turns uric acid into allantoin 1 - uricase of bovine liver 2 - uricase of Aspergillus flavus 3 - uricase of Candida utilis 3 4 activity [jjkat.l"1] ways of measuring enzymes and substrates including calibration catalytic activity concentration of enzymes : analytical approach must be kinetic : reaction according to zero order kinetics and maximal rate Vmax constant concentration method : out of time necessary to reach specific concentration change constant time method : out of signal intensity reached in specific time constant sample 1 with higher and 2 with lower activity vertical lines : constant time method horizontal lines : constant concentration method time 180 Z1 / 2 & to (D constant concentration cn 'to calibration enzyme as analyte - definition of catalytic activity concentration of enzyme by means of product/s/, which is/are created by enzymatic transformation of respective substrate or by measuring its decrease enzyme as analytical agent - calibration is done using standard solution used instead of sample calibrants : certified reference materials(CRM) : serum calibrators (lyophilised sera enriched by respective analytes), their content is determined by definitive or reference methods, if available, or using primary standards 181 brief principles of enzyme analytics constant temperature of incubation mixture + 0.1 °C (37 ±0.1 °C) enzyme determination: incubation mixture - sample (enzyme), buffer and other needful components of reaction; started by solution of thermostated substrate : starter volume not >l/20 to 1/10 of total volume : substrate must be in sufficient surplus > ca 20x Km : starting also by sample (e.g. serum) substrate determination: as in 2), starting by sample catalytic activity concentration of enzyme determination: calibration as within definition of catalytic activity concentration of enzyme by means of product, which is created by enzymatic transformation of respective substrate or by serum calibrators and CRM substrate enzymatic determination: calibration by standard solution of substrate, or CRM and serum calibrators 182 optimisation of enzymatic approaches reaction mixture - multiple components mutually influencing each other, optimisation by so-called relaxation method (SVA) : gathering data on enzym(s) including Km (estimation sufficient) : searching for main parameters of enzyme reaction by MVA optimisation approach method - validation =^> fulfils for given aim the analytical and clinical demands 183 DNA analysis in clinical biochemistry oncology cancer diagnostics presence of mRNA of marker proteins: mRNA tyrosinase (melanoma), GAPDH (lung cancer), E-cadherin (intestines), AIBl receptor (pancreas), hypermethylation of DNA (prostatic cancer) prenatal diagnostics inherited metabolic or neurodegenerative disorders galactosemia, phenylketonuria, Wilson disease, MCAD (disorder of ß-oxidation of fatty acids), Friedreich ataxia, fragile X chromosome syndrome etc. legal medicine identification by means of so-called genetic fingerprints kinship identification; paternity, maternity, victim or culprit identification 184 infectious diseases proof of etiological agent : virus (HIV, cytomegalovirus, hepatitis viruses B, C, enterovirus, adenovirus, herpes simplex virus) : bacteria {Mycobacteria, esp. M. tuberculosis, Chlamydia trachomatis, Bordetella pertussis, Borreliae, Neisseria gonorrhoeae, Legionella pneumophila) : fungi and parasites (Candidae, Aspergillus, Leishmania, Plasmodium, Trichomonas) terrorism- anthrax, tularaemia, botulism etc. monitoring of post-transplantation states monitoring of patient state after transplantation level of cytomegalovirus and herpes viruses, DNA-chimerism food quality monitoring and veterinary medicine pathogen monitoring mycobacteria, echinococci, rotaviruses, PRRVS and BVDV viruses within stock animals, helicobacters in milk, Escherichia coli 0157 mostly in beef or enteroviruses generally in food and drinking water 185 DNA structure primary structure - sequence base = purines, pyrimidines nucleoside = base + (2'-deoxy)ribose nucleotide = nucleoside + 5'-phosphate MP - monophosphate, DP - diphosphate, TP - triphosphate N K O N H. HN O H 7 ^ 9. 7,s> N' N N3 N H lil HNt O ■CH, adenine (A) 5' HOCH, guanine (G) cytosine (A) thymine (A) NH„ CH2 0 OH N O -R OH H adenine (A) HO-P=0 5'CH, H O base C HOCH2 _0 0 H H OH H H O H HO-P=0 deoxycytidine (dC), R = H deoxycytidine B'-S'-diphosphate 5-methyldeoxycytidine (mdC): R = CH3 (dCDP) 186 secondary structure - DNA double-helix base pairing (Watson-Crick) G-C A-T HO-PO 5 CH2 Q 4'I\H base o3' I HO-P=0 0 H CH base 3-° H H 0 1 HO-P=0 0 I CH base 2 O 0 I H CH, HN-H^°Y^ Y 0 N" -N' cJR adenine-thymine ^HN N < N dR O* I H ^ O guanine - cytosine other base pairing Hoogsteen, Wobble, reversed Watson-Crick mismatch - sequences not matched G-A G-T A-C 187 ssDNA - single stranded DNA dsDNA - double stranded DNA double-helix - forms helix A B dsDNA OWWWW&/ chromatin 30n inch romati n extended chromatin condensed chromosome mmmmimm mitotic chromosome 50 OOOx shortened 2*nm J 1 1 nrn i í 30 nrn T 300 nrn 1 T ?00 i 700 nm T 1400 nm i DNA tertiary structure supra-helix : topoisomerases i- : histones 189 transcription; DNA => m RNA translation; m RNA => protein CYTOPLASMA free amino acids m RNA translation /YjyQQQgOťtQ^ ribosome mutation DNA A DNA sequence, A structure and number of chromosomes RNA A gene expression, RNA of errorneous sequencie genealogical investigation disorders diagnostics symptom, i^ phenotypic manifestation, respectively protein A enzyme activity, disorders in cell and tissue structure molecular-biological investigation A organ structure and function, metabolism disorders, developmental disorder 191 analytical approaches >ÍXl_r íl li 11 11 H li ü V? ji iL iL li íl II il U k+ It It If ri it 11 ju. xf J_I_L ■ M li ti ■ * k numerical aberration monitoring : monosomia (Turner syndrome), polysomia (Down syndrome) identification of known sequence : mutations (phenylketonuria) identification of unknown sequence : sequencing of new pathogens TTCCAGTCG-GG AMC CTGTC GTGCCAGCTGCATTAAIGA ATCGGC CA AC GCGCÜGGGA GAGGC at 250 260 270 2SD 290 1Ú0 'A Ä A GC CTG GGGTGC CTAATGAGTGA GC TÄ ňC TCACATTAAT TGC GTTGC G C TCAC TG C CC GCT IM 150 200 118 220 230 240 MáJtiÚúÚéMidAáikm m^i0MMáJmáím.ák JTTTGC GTATTGGGCGCTCTTCC GC TTCC TCGC TCAC T G AC T C CC TGCGCTC GGTC GTTC GGC TG 360 !}■?£ wiJĚm Mfe DNA state monitoring : methylation: deoxycytidine (fragile X chromosome syndrome) basic approaches of NA analyses : PCR amplification :: multiplication of original DNA, 25 cycles ~ 224= 17 xlO6 copies : cleavage by restriction enzymes :: fragmentation of amplified DNA (specific) :(blotting after gel electrophoresis) :: separation of particular DNA sequence : hybridisation - on blotting membrane or on chip Cj :: reaction with complementary labelled ssDNA ^úĹ'J 193 DNA isolation PCR: 10 - 500 |jg of human DNA 1 - 10 |jg of bacterial DNA 0.1 - 1 |jg of plasmid DNA 1) NA extraction + sorption on Si02 after cytolysis 2) gradual soft degradation of cells by organic solvents sample preparation and separation of DNA affinity purification - based on hybridisation abilities of NA : sample + lysation buffer; + biotinylation buffer; incubation : after incubation =^> microtube with avidin or streptavidin modified surface adsorption on silikagel : NA in presence of guanine adsorb on silikagel : elution by A pH and ionic strength gel filtration : micro-column filled with gel : adsorption on silikagel and gel filtration might enhance centrifugation 194 polymerase chain reaction original DNA 1st cycle 2 molecules sHi ľ (=15 target sequence / \ l)a2) 3) 4) 3-t ľlS A • primers - I. . I V ; ■o .: /i i\ 2nd cycle < 4 molecules 3rd cycle 8 molecules I 5) DNA cleavage by restriction endonuclease =^> =^> GELFO separation =^> blotting =^> hybridisation =^> label visualisation DNA - Southern blot i 2 3 4(L)5678 910 1 234(056789 10 1 2 3 4©5 6 7 8 9 10 hybridisation ssDNA fragment (analyte) + + labelled complementary ssDNA fragment (probe) : chromophore, fluorophore, radioactive isotope (32P) cytogenetic diagnostics FISH - fluorescence in situ hybridisation monitoring polysomia, translocation, inversion, deletion possibility to analyse interphase chromosomes : does not need to cultivate cells and prepare metaphase chromosomes complementary pairing of investigated DNA with fluorescence labelled probe sequence of investigated gene : individual gene : whole or part of chromosome or centromeric or telomeric regions detection - fluorescence microscopy 199 mFISH - multicolour FISH high resolution banding combination of more fluorochromes and of more probes SKY- spectral karyotyping identification of numeral and structural chromosomal aberrations complementary pairing - 55 fluorescence labelled probes computer imaging and analysis 200 CGH - comparative genomic hybridization visualisation of chromosomal disorders parallel hybridisation of two DNA samples labelled with different fluorochromes : DNA of patient (tumourous), labelled green : control DNA (DNA of healthy person), labelled red gene therapy transfer of genetic material with therapeutic effect (AW; adenovirus vector) targeted knock-out of aberrant protein (cancer; antisense therapy) renewal of mutated gene expression (inheritable diseases) problem of cellular defence mechanisms to foreign DNA (transplantation) problem of selective defect cells therapy proteolysis . antisense DNA w/gene mRNA \ a RNAse destruction translation f tfb&teXir. -*+ mRNA complementary double-helix target protein 202 colourness physical phenomenon 1 J IX. indication => sight => colour wavelength (nm) 700 650 600 550 500 450 400 j______i_______i i ii red yellow blue J.UV orange green violet blue green-blue blue-green violet red. a violet red orange green green. yellow yellow sunlight - white, colourless decomposed in prism = refractive index is function of wavelength : more inclined from straight line of original ray are violet rays, less yellow ones human eye percepts as colourful rays those with wavelength 400 to 760 nm if the object absorbs yellow] it reflects blue =^> appears to be vice versa and so-called complementary colours 203 absorbance description Bouguer-Lambert-Beer law 1 = 1 * 10 -£*c*d concentration c[mol/l], molar absorptivity e, thickness of abs. layer d [cm] layer transparency: I / I0 absorbance A [l/mol.cm]: A = log I0 / I = c*c*d £= A / c*d =^> proportional to number of molecules of absorbing substance spectrophotometric absorption curve (absorption spectrum) A = f(k) o.s>- u c (O .Q o (A (O 0.6- 0.3- 0.0- 450 500 550 eoo 650 700 wavelength [nm] shifts of absorption maximum: absorption spectrum: magon with Mg(II) ions 1 - magon 2 - magon + 2.10"6 Mg(II) 3-magon + 1.10-5Mg(II) 4-magon + 8.10^Mg(II) bathochromic hypsochromic hyperchromic hypochromic : to higher wavelengths : to lower wavelengths : absorbance increase : absorbance decrease if the spectrum changes indiscreetly ^> existence of isosbestic point \all colour components of the same molecule have in it the same absorbance 205 theory of colourness absorption of light by molecules molecule = X atoms (X>2) absorption of light - valence binding electrons : the stronger the bond is, the greater the rate of their oscillations is molecules absorb: light with frequency « to frequency of valence electrons total inner energy of molecule E: E = Erot + Evibr + EeUiCtr o electrons - higher energy of transition to higher energy level =^> =^> do not take part in light absorption in Vis n electrons - lower energy of transition to higher energy level =^> =^> may absorb even in Vis intrinsic cause of colourness: light energy absorption by n electron transition between levels with different E 206 structures causing colourness i chains of conjugated double bonds excitation energy Crl2=Cri2 CH2=CH—Crl=Cri2 ca 180 kJ/mol ca 150 kJ/mol ethane ethyne butadiene lycopene (11 conjugated double bounds) CH3 | CH3 CH3 CH3 1 CH3 1 3 CH3 C=CH-CH=CH-C=CH-CH=CH-C=CH-CH=CH-CH=C-CH=CH-CH=C-CH=CH-CH=C 1 1 1 CH2 1 CH2- /CH3 CH=C l CH2 H3\ 1 ^C=CH-CHo A = 155 nm A = 190 nm A = 210 nm A = 506 nm addition effects substituents\\ nudeophilic, electrophilic :: charged oxidation I reduction planarity of structure complexation with metal ions detergents', molecular compounds 207 substituents - nucleophilic, electrophilic substituents - charged influence the electronic distribution nucleophilic: : amino group (free electron pair) : hydroxyl group (two free electron pairs) benzene Amax = 255 nm > phenol Amax = 275 nm > aniline Amax = 282 nm electrophilic: : nitro group, carbonyl group and imino group nitrobenzene Amax = 268 nm, acetophenone Amax = 279 nm :: combination of nucleophilic and electrophilic group: bathochromic shift ionisation enhances the effect of substituen t presence nitrophenol pKd = 7.16 : acidic medium colourless Amax = 315 nm (left) : alkali medium yellow (middle) : quinoid structure (right) Amax = 404 nm 208 OH I Ol 0=N=0 0=N=0 0=N-0 Oxidation / reduction oxidation increases number of conjugated double bonds CI 1 ľ 0= £- --o -OH + 2H + + 2e" HCT 9 -NH- ■O -OH 1 CI 1 CI 2,6-dichlorophenolindophenol - blue reduction - leucobase (colourless) reduction rarely enhances absorption H H H LNH 2«- 0 II .C. ~NH' isľ NADH NAD coenzymes derives from nicotinamide CH3-CH2-OH + NAD+ -> CH3-CH=0 + NADH + H+ 209 structural planarity free overlapping of n- electrons in conjugated system = planar structure Pr Me V Me jnase + 02 | heme oxygenase - C02 - Fe(II) heme - red MeV MePr Pr Me Me V H H n H biliverd in reductase _ + NADPH 1 NADP+ Pf Me Me V Me V Me Pf Pí Me Me V H H H H biliverdin - green bilirubin - yellow metal chelates creation of complex {ML} and of co-ordination bond at expense of free electron pair included into system of conjugated double bond is followed by enhancement of colourness l-nitroso-2-naphtol : yellow-orange : Fe, Ni or Cr ions :: green and brown complexes detergents alkalic xylenol orange 2.10 5 mol/l 1 - XO, pH 10.5 2 - XO and 5.10^ mol/l CPB pH 10.5 3 - XO, pH 6.4 molecular compounds : aromates, heterocycl. bases &arom. compounds with nucleoph. substituents : aromatic hydrocarbons with electrophilic substituents quinhydrone 2: 0.6- u fü 0.4-i_ O to 1 / \ / 3^2\ ro 0.2- n D- 400 450 500 550 600 wavelength (nm) 650 use of colourness in clinical diagnostics acido-basic indicators pH determination : alkalimetric titration => determination of total protein by Kjeldahl method (NH3) : orientational pH determination => semi-quantitative determination of urinal pH - mixed indicators (continuous transition) analyte determination : semi-quantitative determination of serum albumin - protein error : semi-quantitative urea determination - urease reaction, NH3 production : microbial contamination indication - process of sugars into organic acids 212 redox indicators determination of reducing/oxidising substances : determination of ascorbic acid (vitamin C) : oxidative copulation - glucose determination, uric acid, cholesterol : ELISA determination with POD (horse-reddish peroxidase) : AST, ALT enzymes determination (phosphatases) using secondary enzymatic redox reaction orqano-aqents and metallochromic indicators metal ions determination - CI (I), Ca(II), Mg(II), Cu(II), Fe(II) chlorides - titration Hg(N03)2; Hg(II) + diphenylcarbazon - blue-violet colour calcium - arsenaze III, o-cresolphthalein complexon magnesium - magon, calmagit copper - bathocuproine iron - bathophenanthroline, ferrozine copulation agents determination of reacting substances azocopulation => : azo-dyes - conductive connection of conjugation chain of double bonds of original molecules by azo-group =^> planary structure of new molecule : aryl diazonium cation as electrophilic agent : activation of/^-position of phenol by dissociation of hydroxyl hydrogen C6H5- H= Ň+ + -C6H5-R -► C6H5-N=N-C6H4-R reaction of bilirubin with diazosulphanilic acid 214 oxidative copulation => : molecule with two aromatic rings conjugated through imino-group : new molecule has quinoid structure : further substituents increase its polarisation =^> highly colourful use of created hydrogen peroxide C6H5-R + 2 H202 ^d> 0=C6H4-R + 3 H20 R-NH2 + 0=C6H4-R -> R-N=C6H4-R + H20 -R is =0, -NH2 non-chromogenic products are transformed into colourful 1-naphthyl phosphate ^> 1-naphtyl + copulation ag. => colourful product acidic phosphatase 215 redox agents determination of reacting substances oxidation => transformation of reduced leucoforms into colourful H3C CH, H3C C^ h2"n^ y \ V-ŇH2 + H5O5, POD HŇ=( \ =/ \=NH -2^0 ^C C^ H3C CH^ H¥-([ V -Cr- +H+ H2i\T—/ y nf 7—Ň+H2 -1+ HjC CH: H^C CH3 3,3',5,5-tetramethylbenzidine and oxidation to blue : oxidation intermediate - semi-quinone chromoqenic substrates for enzyme reactions colourful them-selves, or by enzymatic transformation created colourful product alk. phosphatase 4-nitrophenyl phosphate ^> 4-nitrophenol 312 nm 404 nm glutamate transferase L-Y-glutamyl-3-carboxy-4-nitroanilid gmi> 5-amino-2-nitrobenzoic acid NH2 NH: A OH" **-------*- (l1 ^f-coó pHB V ■^coo" 1 o=h J-O" 217 biomolecule labelling : affinity- weak interactions : covalent label types affinity : peptides/proteins : NA : chromophores, fluorophores covalent modification : chemical : enzymatical h"o -VŠ R2 trypsin ----------> 218 : isotopic (radioactive, heavy) : chromophores, fluorophores, luminophores : enzymes IB OH + H ° R2 Rt is label R2 is biomolecule RrCO-OJM, Ö ť + R2-N^ succinyl innide ester RrN=C=S +P^-N^ isothiocyanate R^-CO-NHP^ + HO^J Ö carbox amide RrNH-CS-NH-F3 thiourea FVSCfe-CI + RrNr^ -----»• RrSOrNH-R2+ HCl sulphonyl chloride sulphoamide O R.-N. # + R 2-^1 Ri-N 0 rnalein irnide # (T *S-R, thinfithfir medical microbiology diagnostic approaches microorganisms - basic component of biosphere : pathogenic - health dangerous : occasional pathogenic - dangerous only sometimes {Escherichia coli) medical microbiology - microbiologic analysis proof of etiologic agent - infection progenitor; its sensitivity to antibiotics and chemotherapeutics medically important microorganisms : bacteria : microscopic fungi : protozoa : viruses : subviral agent (prions) 219 general approach in microbiologic diagnostics : clinical investigation of patient : collection and transport of sample : evidence and formulation of approach 1st series: microscopy, antigen and DNA/RNA detection 2nd series: isolation of agent, event, indirect proof 3rd series: identification of agent 4th series: determination of sensitivity on antibiotics : diagnosis and therapy direct proof: microorganism finding microscopically - shape, staining immunologically - proof of pathogenic antigens genetically - sequencing biochemically - proof of specific pathogen metabolites indirect proof: immunologically - finding of antibodies against pathogens microscopic proof : fast and cheap : less sensitive - from concentration 105/ ml native preparation : proof of larger microorganisms - some parasites and coetaneous fungi :: syphilis diagnostics [Trepomena pallidum, treponemata) : mycologic preparations - „native" only in technical sense; stained by Parker ink or fluorescence dye stained preparation fixation - disruption and adhesion of microbial cells on microscopic slide heating of the overlay over flame or denaturation by chemical agents (regardful; methanol, protein antigens - ethanol, viruses - acetone) : orientational simple staining - methylene blue : diagnostic staining (differential) - staining according to Gram, Ziehl- Neelsen, Giemsa and fluorescence staining approaches 221 Gram staining : presence of microorganisms (significant number) : size, shape (cocci, bacilli...) : mutual arrangement (diplo-, staphylo-, strepto- etc.) preparation background - presence and view of macroorganism cells (epithelia or leucocytes) and other structures (mucous fibres etc.) divides microbes into blue-violet coloured gram-positive microbes pink to red coloured gram-negative microbes procedure: fixed preparation for 20 s into solution of crystal violet; then for 20 s in iodine solution, alcohol washing (max. 20 s), water washing, immersing into saphranine solution and final water washing structure of bacterial wall- complex of crystal violet with iodine is within gram-negative bacteria easily washed out by alcohol; they could be then easily stained pink by saphranine or diluted fuchsine diagnostics of purulent affections (meningitis, gonorrhoea, anaerobic infection, inflammations) 222 Ziehl-Neelsen staining bacteria unstainable by Gram procedure {Mycobacterium tuberculosis) procedure: fixed preparation is stained while steam heating in carbolfuchsine, destain by acidic alcohol and re-staining with e.g. methylene blue; acido-resistant bacilli are stained pink, preparation background is blue fluorescence staining : more sensitive proof of acido-resistant bacilli procedure: heat fixed preparation id stained by mixture of fluorescence dyes auramine and rhodamine, differentiates by acidic alcohol, re-staining by fuchsine; on dark crimson preparation background brilliant yellow bacilli Giemsa-Romanowskv staining : in haematology serves to stain smear blood; in microbiology to prove protozoa (malaria, trypanosomes, leishmania, trichomonas), poorly Gram stained bacteria (ehrlichia, rickettsia etc.) and to picture viral inclusions procedure: fixed by methanol and stained for 2 h by Giemsa dye (1:10 water diluted). Giemsa dye - azure with eosin; bacteria are stained dark blue, nuclei of protozoa lake, their cytoplasm light blue ™ isolation, cultivation of microorganism =^> direct evidence procedure : on cultivation media : bacteria, yeasts, mould and protozoa : in cell cultures {in vitro, in vivo) : protozoa and intracellular parasites cultivation media basic liquid medium (bouillon) : meat extract :: inoculation by loop, incubation in thermostat basic solid medium (nutritive agar) : algae boiled in bouillon - gel (agarose, agaropectin) :: inoculation by loop - cross smear, incubation in thermostat enriched media : growth factors (vitamins, AA, nucleotides) : blood agar, egg media {M. tuberculosis) selective media : growth inhibitors for unwanted organisms - selection 224 diagnostic media : growth factors, selection inhibitors, substrate and indicator End medium (enteric bacilli), MacConkey medium media for antibiotic sensitivity determination : growth factors, selection inhibitors, substrate and indicator Mueller-Hinton agar transportation media : without nutrients, wet, metabolism inhibitors :: 24 h of survival Amies medium - inorg. salts, sodium thioglycate, active carbon, 0.4% agar in vitro cellular cultures : chicken embryos - viruses, ricketsia, chlamydia tissue cultures, monolayers under nutritive solution (Eagle essential minimal medium) in vivo : guinea-pigs, mice „3 FT rules : refinement - carefully prepared experiments under best conditions : reduction - lowest possible number of animals : replacement - try to evade the vivisection by other approach, e.g. by tissue cultures or genomics tuberculous mycobacteria - guinea-pigs; rare and unrepeatable samples (liquor, exempt lymph nodes etc.) vivisection is more sensitive than cultivation- tularaemia or psittacosis virology - laboratory mice; tick-borne encephalitis virus isolation, coxsackieviruses (velamentum and hearth muscle inflammation) proofs for microbial toxins - not possible without vivisection 226 .. .. .. biochemical proof diagnostic media ------------------K------ saccharide media - ability of microorganism to metabolise certain saccharide product =^> acid i pH of medium - acido-basic indicator adonitol, arabinose, cellobiose, galactitol, fructose, galactose, glucose, inositol, inulin, lactose, maltose, mannitol, mannose, melesitose, melibiose, rafinose, rhamnose, ribose, sucrose, sorbitol, starch, trehalose and xylose substrate media - ability to enzymatically proceed the substrate deamination - phenylalanine and tryptophan decarboxylation - arginine, lysine and ornithine hydrolysis - urea, hippurate and tributyrine reduction - nitrates to nitrites metabolic media - take-up of certain substances citrate, acetate or ma late - sources of carbon : growth factors auxanoqram - determination if yeast grows not better in vicinity of tablet with certain saccharide 227 used microbial enzymes: N-acetyl-ß-D-glucosaminidase (NAG), C8-esterase, a-galactosidase (aGA), ß-galactosidase (ßGA), ß-glucosidase (ßGL), ß-glucuronidase (ßGLR), yglutamyl transferase (GGT), leucyl aminopeptidase (LAP), pyrrolidonylarylamidase (PYR), urease and ß-xylosidase (ßXY) immunodetection agglutination on glass-plate : antigens of corpus and flagellum Salmonella enteridis, citrobacteria, pseudomonades, Vibrio cholerae, bordetella, meningococci viral identification virus-neutralising test : specific antibody inhibits some biological effect of virus 228 antibiotic sensitivity antibiotics, synthetic anti-microbial chemotherapeutics qualitative proof disc diffusion test principle: around the disc made of filtration paper saturated with antibiotics, sensitive microbe will not grow - inhibition zone of certain diameter is created strain sensitive towards given antibiotics - zone is same or larger than within reference strain tested antibiotics - depends of microbe species, disease characteristics and sample type, of which the microbe was cultivated and on local situation in development of microbial resistance towards antibiotics 229 quantitative determination dilution approaches (dilution of antibiotics); minimal inhibition concentration (MIC) of given antibiotics for evaluated microbial strain : microtitration plate 8x12 with defined concentration of certain antibiotics decreasing in geometric order : after inoculation and incubation we check, if bouillon remained transparent (total growth inhibition), or if there is precipitate or sediment MIC - lowest antibiotics concentration able to stop growth; |jg/ml, mg/l minimal germicidal concentration - lowest antibiotics concentration able to kill examined strain : increasing concentration in series especially today - resistance of microbes towards antimicrobial substances antibiotics resistance - change of target molecule, worsened penetration into cell, increased excretion of antibiotics from cell or appearance of enzyme, which inactivates antibiotics 230 proof of microbial component __________________________________________________________ indirect proof traces, laid during the infection by pathogen in organism : microbial antigens, toxins, metabolic products and typical NA sequences in majority of cases it is proof by means of antibody (Ab) amount determination serologic test immunoassay syphilis, glandular fever, HIV infection etc. : proof of significant increase of Ab amount; Ab amount is called titre 231 case study method development for clinical diagnostics J determination of alkalic phosphatase alkalic phosphatase (ALP) - analyte relevant to liver function, growth of bone apparatus, placenta etc. 4 main isoenzymes: osseous, hepatic, enteric and placental case study - typical case for analytical method development : influence of method, temperature, buffer, modifiers, estimation of optimal substrate concentration, approach for reference interval determination + kit manufacture and way of individual agents stabilisation 232 basic properties of ALP ^___ d wi Ol ^ ■ Ol ^ ■ ortophosphoric-monoester phosphohydrolase with alkalic optimum : catalyses hydrolysis of phosphate monoesters to inorganic phosphate and respective alcohol : it may also hydrolyseaW types of compounds with bonds P-O-C, P-O-P, P-S and P-N, with exception of compounds with P-C bond : in case of phosphate monoesters it may also transfer phosphate group hydrolysis R-0-PO-(OH)2 + H20 ^p> R-OH + H3P04 transphosphorylation R1-0-PO-(OH)2 + R2-OH ^p> Rj-OH + R2-0-PO-(OH)2 efficiency of transphosphorylation depends mainly on type and concentration of acceptor 233 ALP : metalloprotein; co-factor Zn(II) (4 atoms) : Zn(II) plays important role within transphosphorylation reactions of ALP : Co(II) supports hydrolysis, but not transphosphorylation non-specific inhibitors: EDTA, KCN, cysteine, o-phenanthroline, 8-hydroxyquinoline-5-sulphonic acid etc. : removing first 2 Zn atoms, the enzyme activity decreases in about 90% : remaining 2 atoms are more difficult to remove; results in completely inactive apoenzyme specific inhibitor - L-phenylalanine activators: Co(II), Mg(II) and Mn(II) ions, whereas Be(II) a Zn(II) act as inhibitors 235 ALP importance discovery: 1907 in rice germs analytical determination and use in diagnostics : 1926 - diagnostics of bone disorders : 1930 - obstructive jaundice serum of healthy contains mostly liver and osseous isoenzymes T activity of ALP : growth disorders a some osseoms (osseous isoenzyme) : hepatobiliary system disorders (cholestase and tumour metastases into liver) ^ activity of ALP : hypothyreosis (cretinism), scurvy, irradiation disease, heavy anaemia and within immunosuppressive medication season variations: UV-light influence; in winter (low sun radiation) t ALP, in pregnancy t ALP in about 12 to 50 % (placental isoenzyme) 236 ALP determination methods catalytic activity: depends on substrate and reaction conditions - type, pH and concentration of buffer, temperature, presence of modifiers history: 1926 - substrate hexaphosphate 1929 Kay - substrate ß-glycerolphosphate, without buffer; determination of inorganic phosphorus (48 h!M) later - glycine buffer pH 8.8, phosphorus by phosphomolybdate blue (3 h) - barbitate buffer pH 10.8 new substrates: phenyl phosphate and 4-nitrophenyl phosphate, Phenolphthalein mono- and diphosphates, thymolphthalein monophosphate, 3-O-methylfluorescein phosphate, naphthyl-AS-MX-phosphate, methylumbelliferyl phosphate, indoxyl phosphate etc. increasing sensitivity - phenyl phosphate, determination of released phenol by oxidative copulation with 4-aminoantipyrine and ferricyanide noval - chromogenic substrates: phenophthalein phosphate : thymolphtalein phosphat 237 Substrate : unambiguously defined, sufficient chemical purity, must have hydrolysis product with auto-indicative properties today - 4-nitrophenyl phosphate (NPP 1), is cleaved into phosphate and 4-nitrophenol (NP 2) NP is acido-basic indicator with dissociation constant pKa = 7.16 concentration of NPP: 5-20 mmol/l temperature ALP - at 37 °C faster inactivation ■I 3 37 °C 30 °C T-----------------"- 3D «Jörne [min]» absorbance 0j6 0.3 OjO 260 300 360 400 450 500 wavelength [nm] 238 pH and buffer already not used buffers: barbitate, glycine, hydrogen carbonate, 2-amino-2-methyl-1-propionic etc. ALP activity is strongly influenced by buffer : buffer is simultaneously also second substrate (phosphate acceptor) within transphosphorylation activity influencing by buffer : neutral / inert (carbonate, barbitate etc.) : inhibiting (glycine, propylaminic etc.) : activating (transphosphorylating buffers) buffer for ALP phosphate acceptor sufficiently stable available in p.a. purity dissociation constant pKa ~ 10 H,N H C H, H, )H H2C -NH ;h, ;h. H, H.- ;h. EAE ethylarninoethanol N H, H,C CH -C—OH H, AMP was recommended by IFCC AMP 2-amino-2-rnethyl-1-propanol 239 DEA diethanolarnine H2C—+1 —CH3 2 I H 3 HC—OH R MEG N-rnethyl-D-glucarnine R- glucosed ways of measurement before: enzyme activity kinetically, manually and discontinually (two-points) today: fast kinetic continual approach modifiers of ALP inhibitors: compounds of arsenic, phosphates, substances able to bind zinc ions stronger than enzyme : non-specific chelatogenic inhibitors: EDTA and KCN specific activator: sodium ion in MEG buffer 240 ALP determination in MEG buffer optimisation of ALP determination : literary research (properties, determination methods etc.) : demarkinq orientation conditions of analytical approach (buffer type and its pH and concentration, type and concentration of substrate) : optimisation of incubation mixture composition exclusively with NPP substrate buffer : MEG - good transphosphorylation properties, low reactivity, high purity and ready availability : DEA and AMP excluded for content of inhibiting impurities 241 reaction conditions and work-f low temperature 37 °C wavelength 420 nm pH (37 °C) 10.1 ±0.1 opt. path length 1 cm MEG buffer 0.35 mmol temperature (37.0 ± 0.1) °C NPP substrate 15 mmol/l signal AA sodium chloride 70 mmol/l measurement interval 30 - 120 s magnesium chloride 0.5 mmol/l serum/incub. mix (v/v) 1:51 (0.0154) chemicals purity NPP: < 0.05 % free 4-nitrophenol, < 0.25 % free inorg. P043" molar absorbance in 10 mmol/l NaOH A = 311 nm - 9 857 + 751 l/mol.cm MEG: melting point 129 - 131 °C NP: molar absorbance in 10 mmol/l NaOH A = 401 nm -18 380 l/mol.cm 242 stability of agents buffer - at least 1 month at +5 °C (without bacterial contamination) substrate - prepare just before use; in dark and cold at +5 °C stable at least 24 hours standard solution of NP - in dark and cold stable at least 1 month calculation of catalytic activity concentration fS-ALP [|Jkat/l] = AA420 * F F is factor, AA420 = (AAj - AA2) absorbance of reference solution (AA2) - compensates absorbance of NP created by spontaneous, non-enzymatic decomposition of substrate due to alkali medium presence factor F: 1) from calibration solution without ALP 2) using molar absorbance of 4-nitrophenol 243 optimal conditions of ALP determination activity 4 3 2 1 activity osseous ,__------Ü----------0_ hepatic enteric 2 -i-----------1------------r- 8.3 9B 9.9 10.2 10.5 pH influence PH reference interval 00 0.2 0.4 06 09 MEG concentration [mol.ľ1] influence of MEG concentration activity osseous * NPP + serum hepatic enteric -a D B 10 15 20 NPP concentration [mol.ľ1] influence of NPP concentration fS-ALP [pkat/l]: males 0.90 - 2.20, females 0.74 - 2.10, children 1.20 - 6.30 200 adults, 112 boys and 152 girls repeatability and reproducibility repeatability: examined in 10 external laboratories, precision in series and day-to-day; N = 20; 0 rel. standard deviation: 3.5 % manual analysis; 2.5 % analyser reproducibility: in 5 laboratories; 1.4 - 4.2 % 244 kit for ALP determination 1 agent 1 (6 vials) : solid substrate, finally for NPP solution of 1.12 mmol/vial : substrate solution is prepared by dissolution of one vial content in 5.0 ml of distilled water; in dark and cold at +5 °C, the solution is stable ca 1 week agent 2 (300 ml) : MEG buffer in liquid state, concentration 0.56 mol/l, which contains as conserving agent 5-bromo-5-nitro-l,3-dioxane 0.3 mg/l and N-methylisothiazolon 1 mg/l agent 3 (2 ml) : standard solution of NP in ampoule under N2 containing NP at 2.4 mmol/l and Na2EDTA 0.05 g/l storage: in dark and cold at 2 - 8 °C, stable for 24 months working procedure: mutual voluminal ratios of serum, buffer and substrate are 1:50:5, so 0.02 + 1.00 + 0.10 ml 245 importance of buffer in bioanalysis buffer - reaction medium/environment : keeping pH (optimum) : secondary substrate (enzyme reactions) : modifiers - inhibitors, activators 1900, Fernbach and Hubert: partially neutralised solution of phosphoric acid serves as protection against abrupt changes of solution alkalinity or acidity within research of enzyme amylase buffer choice basic characteristics: dissociation constant of acid (part of buffer); pKa dilution influence, ionic strength, buffer capacity and pH temperature influence pH range: pH = pKa + 1, while buffer is the most effective at pH = pKa 246 compatibility and synergic influence of buffer compatibility chemical : good water solubility (bad with barbiturates) : non complexing for metal i. - Ca(II), Mg(II), Mn(II), Zn(II), Fe(II)/(III) etc. : soluble (so mostly no carbonates, phosphates, citrates) : non-reactive with other components (proteins, saccharides + borates) : invulnerable to bacterial contamination (so no phosphates, citrates etc) biochemical : influencing activity of some enzymes (phosphates inhibit phosphatases, carboxylases, phosphoglucomutases etc) : interference in processes of oxidative phosphorylation (barbiturate) : antisepticity; activity blocking of most of enzymes (phenols) : ampholytes (zwitterionic) oxidised by flavin mononucleotides (BICIN, TRIS) : reactive and inhibiting buffers (TRIS) synergicity modification - activator (GlyGly; fibrinogen) or secondary substrate (MEG) 247 main bioanalytic buffers abbrev composition P*a M ES 2-(N-morpholine)ethane sulphonic acid 6.1 BIS-TRIS 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-l,3-propandiol 6.5 BES N,N-bis(2-hydroxyethyl)-2-aminoethane sulphonic acid 7.1 HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethane) sulphonic acid 7.5 TRICIN N-(2-hydroxy)-l,l-bis(hydroxymethyl)ethylglycine 8.1 BICIN N,N-bis(2-hydroxyeteyl)glycine 8.3 AMPSO 3-[(l,l-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropane sulphonic a. 9.0 CAPSO 3-(cyclohexylamino)-2-hydroxy-l-propane sulphonic acid 9.6 CABS 4-(cyclohexylamino)-l-butane sulphonic acid 10.7 248 analysis of selected analytes inorganic, organic analytes and bioanalytes ammonium content: degradation of amino acid in liver; toxic (CNS) =^> urea determination: in plasma; reference interval 11 - 35 pM chemical methods procedure: ammonium is alkalised, separated and absorbed into acidic solution (Conway technique) : titration, conductometry, ISE, Nessler agent (yellow) or Berthelot reaction (reaction with phenol and with alk. hypochlorite into blue quinonimine dye) laboriousand non-automatable enzymatic methods enzyme: glutamate dehydrogenase (GLD) 2-oxoglutarate + NH4+ + NADPH £^> L-glutamate + NADP+ + H20 : absorbance decrease of NADPH at 340 or 365 nm procedure: TEA 150 mM, pH 8.6 + 2-oxoglutarate 15 mM, ADP 1.5 mM, GLD min 800 U/ml and NADPH 0.12 mM 9.c phosphorus, phosphates content: inorganic and organic phosphates; bone tissue, nucleic acids, phospholipids, coenzymes, ATP etc. determination: in serum; reference interval 0.7 - 1.6 mM chemical methods inorganic phosphates. : reaction with ammonium molybdate (colourless phospho-molybdate complex) : with ammonium vanadate-molybdate (yellow complex of ammonium phosphoric vanadomolybdate); hydrolyse partially also organic phosphoric esters and thus makes the determination falsely positive organic phosphates, after mineralisation of denatured proteins; depends on pH (strong acidic medium) procedure: serum deproteination (strong acid), supernatant analysis: measured at 340 nm; phosphomolybdenite blue (after reduction tin(II) chloride, aminonaphthalene sulphonic acid, methyl-p-aminophenol sulphate, iron(II)-ammonium sulphate, ascorbic acid etc.) at 882 nm; vanado-molybdate phosphoric acid at 420 nm enzymatic methods do not catalyse phosphoric ester hydrolysis, no need for sample deproteination : glycogen Phosphorylase, phosphoglucomutase and glucose-6-phosphate dehydrogenase - NADPH : purin nucleoside Phosphorylase, xanthine oxidase (peroxidases) - oxidative copulation : sucrose Phosphorylase - NADH 250 magnesium content: 21 - 28 g in ca 70 kg (60 % bones, 20 % muscles, 19 % other tissues, ca 1 % extra-cellular liquid); cofactor of almost 300 enzymes determination: in serum/plasma; reference interval 0.65 - 1.05 mM chemical methods AAS - optimal; free Mg(II) - ISE : in analysers - photometric methods with calmagit, magon... AAS: procedure: LaCI3 solution (spectral buffer, releases Mg(II) from phosphate complexes and dilutes viscose proteins) concentration 4.3 g/l with 10 ml cone. HCl; sample : buffer 1:50 into flame (acetylene-air), Mg-discharge at 285.2 nm; calibration solution contain interferents Na+ and K+ reaction with magon: procedure: 10 pi serum with 2 ml working solution (20 mM borate buffer pH 9.5 and magon 0.28 mM diluted in mixture DMF and ethanol 5+100), resulting pH ca 11; influence of Ca(II) and heavy metals - cyanide masking + physiologic concentrations of Ca(II), Na(I) and K(I) ions; absorbance decrease of blue complex around 500 nm reaction with calmagit procedure: in partially non-aqueous medium with 2-methyl-2-amino-l-propanol; Ca(II) masking by EGTA, measured at 540 nm 251 hydrogen carbonates content: blood; diagnosis of acidobasic equilibrium disorders determination: in whole blood, plasma and serum as C02 (after acidification); reference interval depends on instrumentation, usually C02 in capillary plasma ca 22 - 31 mM physical methods gaseous carbon dioxide by manometry, laborious and not automatable chemical methods continuous measurement- C02 diffusion through Si-membrane and sorption as HC03~ in alkali buffer, pH 9.2 with Phenolphthalein; colouration decrease (acidification) photometry electrochemical measurement- C02 electrode, partial pressure of dissolved gas in blood enzymatic methods alkalisation - transformation into HC03~ enzymes: phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH): HCO3- + phosphoenolpyruvate hepc> oxalacetate + P043_ oxalacetate + NADH + H+ ^^> malate + NAD+ procedure: alkali buffer ca 70 mM, pH 8 + phosphoenolpyruvate 8 mM, NADH 1.6 mM, microbial PEPC min 17 pkat/l and microbial MDH min 4 pkat/l 10 pi serum/plasma (tempered cuvette) + 1 ml agent, incubation 5 min at 37 °C, absorbance at 340 nm 252 Chlorides content: main extra-cellular anion (67 %) determination: in serum and plasma; reference interval in serum 98 - 107 mM titration: 2 CI" + Hg(N03)2 -+ HgCI2 + 2 N03" procedure: in acidic medium, mercury(II) nitrate solution 5 mM, sample is 0.20 ml of serum, indicator diphenylcarbazon 20 mM in ethanol spectrophotometry. 2 CI" + Hg(SCN)2 -+ HgCI2 + 2 SCN" SCN- + Fe(III) -> Fe(SCN)2+ procedure: measured around 500 nm (red product), range 80 - 125 mM, calibration not linear, linearity by constant amount of Hg(N03)2 which binds ca 60 mmol Ch in 1 L coulometry. Ag+ + CI" -+ AgCI procedure: generating Ag(I) in a constant rate off anode till equivalence, content of chlorides is then directly proportional to measured time; acidic medium (better conductivity) at presence of gelatine or polyvinyl alcohol (reproducibility) 253 copper content: metalloenzymes, in plasma in 95 % on coeruloplasmin determination: in serum; reference interval (pmol/l) 10.1 - 18.4 (males), 11.3 - 25.2 (females) AAS: : only sample dilution necessary reaction with bathocuproin: procedure: deproteination + reduction agent (hydroxylamine or pyrosulphite in combination with p-(N-methyl)aminophenol), centrifugation, supernatant is put into other tube and bathocuproin solution is added; in strongly acidic medium orange complex is measured at 480 nm : depends on glass purity; EDTA, washed by water with ammonium zinc content: metalloenzymes determination: in plasma, serum, saliva, urine; reference interval (serum) 10 - 20 pM spectrophotometry with 5-Br-PAPS procedure: 2-(5-bromo-2-pyridylazo)-5-(N-n-propyl-N-3-sulphopropylamino)phenol at pH 8.6 + making agents (other endogenous metals), at 560 nm, not sensitive enough AAS procedure: sample is 5x diluted by 5% glycerol, measured at 213.8 nm; urine only from 24 h collection, directly into flame; complications - high salt content 254 calcium content: bone tissue (99 %), extra-cellular liquid, free Ca(II) is only active determination: in serum, only free or also bound; reference interval 2.1 - 2.6 mM reaction with o-cresolphthalein complexon: procedure: pH 12 in medium with organic base (2-amino-2-methyl-l-propanol, 2- ethylaminoethanol); measured at 580 nm, 8-hydroxyquinolin suppresses interferences reaction with arsenazo III: procedure: imidazole buffer pH 6, blue complex, measured around 650 nm; specific detergents suppress interferences of proteins AAS: procedure: similarly to magnesium, measured with Ca-lamp at 422.7 nm, expensive, higher precision and accuracy ISE: : Ca(II) activity measured, depends mainly on ionic strength (Na+ and Cl~) - calibrators 255 potassium content: K+ in main intra-cellular cation (4.6 mM) determination: in all body fluids, mostly in plasma and serum, strongly interfered by haemolysis; reference interval in serum 3.8 - 5.2 mM enzymatic methods enzymes: tryptophanase (TR), glutamate dehydrogenase (GLD) tryptophan + pyridoxal-5-phosphate i&í indol + pyruvate + NH4+ NH4+ + 2-oxoglutarate + NADPH + H+ £^> glutamate + NADP+ enzymes: pyruvate kinase (PK), lactate dehydrogenase (LDH) phosphoenolpyruvate + ADP hkí! pyruvate + ATP pyruvate + NADH + H+ ^*> lactate + NAD+ : Na+ concentrate lowering by cryptand chemical methods AES\ procedure: serum diluted by spectral buffer (lithium or caesium, stabilises effective temperature of flame; anion tensides Brij 35 or Sterox SE for better atomisation); flame propane-air; Na, K, Li and Cs with sharp lines at 589 nm, 768, 671 and 852 nm ISE\ : electrode with liquid membrane 256 sodium content: Na+ main extra-cellular cation (ca 142 mM) determination: in all body fluids, mostly in plasma and serum, strongly interfered by haemolysis; reference interval in serum 132 - 142 mM enzymatic methods enzymes: ß-galactosidase (ßGD) 2-nitrophenyl-ß-D-galactopyranosid PGC?Na+> galactose + 2-nitrophenol 2-nitrophenol (chromophore) measrued kinetically at 420 nm, Na+ content lowered to measurable values by cryptand, e.g. Kryptofix 221 chemical methods AES\ as potassium ISE\ electrode with glassy membrane 257 barbiturates content: medical rugs (sedative) determination: in serum (medication monitoring), in urine or stomach (intoxication) chemical methods gas chromatography. procedure: aprobarbital (internal standard) is added to serum, diethylether double extraction, sodium sulphate dehydration and dry evaporation; evaporate dissolved in ethylacetate; GC separation - capillary column (WCOT), flame ionisation detector, carrier gas Ar or He; analysis of different barbiturate types (short, medium and long term affecting) titration analysis procedure: extraction by phosphate buffer pH 7 and chloroform mixture; into organic phase buffer is again added with mercury(II) chloride, extracts and centrifuges; organic phase is titrated by diphenylthiocarbazon (dithizon) solution, change from violet ({Hg(II)-barbiturate} complex) to orange ({Hg(II)-dithizon} complex); hydantoins and cyclic imides of glutaric acid interfere mostly 258 bilirubin and its esters content: metabolism of haeme through biliverdin to bilirubin and conjugates (mono- and diglucuronides esters) determination: in serum and urine; reference interval for total bilirubin in serum (pM): new-borns 68 - 138, children and adults 3.4 - 17.1, out of which conjugated, so called direct bilirubin 0 - 3.4 chemical methods bilirubin and its conjugates determination (and part of bilirubin covalently bound to albumin) is based of azobilirubin creation (acidobasic indicator): red in weak acidic and neutral pH, in strong acidic and alkali range is blue; conjugates (i.e. direct bilirubin) reacts without accelerators; unconjugated bilirubin (i.e. free) does not react, only in presence of accelerators (alcohol, caffeine etc.), which release and solubilise bilirubin from its bonds to albumin determination of total bilirubin at presence of accelerators', within increased content also the direct bilirubin is determined without accelerators; within new-born jaundice and irradiation of new-borns with UV-light, which serves to decompose and remove of free, unconjugated bilirubin, it was observed creation of so-called photobilirubins, which have in contrary to normal bilirubin different reaction kinetics and may distort results of measurements 259 1 4438 57 spectrophotometry with diazotised sulphanilic acid. procedure: IFCC method of total bilirubin determination; sample in cuvette + sulphanilic acid solution in HCl (cleaves bilirubin in methylene bridge) with accelerator solution (mixture of caffeine, sodium acetate and benzoate) + NaN02 solution (diazonium salts); after 10 min in weak acidic medium absorbance is measured of red azobilirubin at 430 -460 nm; determination through blue form is done by adding alkali buffer solution (NaOH and sodium potassium tartrate), and in pH 12 it is measured at 580 - 620 nm determination of direct bilirubin is done without accelerator by stopping diazoreaction by ascorbic acid (diazotisation agent decomposition), it is usually added after 5 or 10 min; total bilirubin is determined with cation-active detergent like accelerator (cetyltrimethylamonium bromide) spectrophotometry after oxidation: procedure: bilirubin oxidation (yellow) by vanadic acid to biliverdin (green); two-point absorbance measurement before and oxidation (3 min); it is possible also to determine direct bilirubin enzymatic methods enzyme: bilirubin oxidase (BOX) bilirubin (yellow) ^x> biliverdin (green) procedure: pH 4.5, measured in range 424 - 465 nm kinetic in time interval ca 5 min; total bilirubin is determined at pH 8.5 at presence of accelerators 260 ethanol content: alcoholic intoxication chemical methods Widmark method; distillation of alcohol off sample, oxidation by dichromate in sulphuric acid or in glacial acetic acid, surplus of dichromate is then determined by iodometric titration; gas chromatography is used in forensic investigations : to prove heavy (chronic) alcoholism, so called carbohydrate deficient transferrin (CDT) test was developed based on heterogeneous immunoanalysis enzymatic methods enzyme: alcohol dehydrogenase (ADH) CH3CH2OH + NAD+ ^> CH3CHO + NADH + H+ procedure: sample deproteination by perchloric acid, alcohol is determined from supernatant in alkali buffer pH 8.7 (pyrophosphate, semicarbazide and glycine), absorbance at 37 °C at 340 nm after incubation 25 min 261 drugs of abuse (DOA) content: psychofarmaca and their precursors - alcohol, amphetamine, barbiturates, benzodiazepines, cannabinoids, cocaine, methadone, opiates, antidepressives, anabolic steroids etc. determination: belongs to point-of-care testing (POCT) chemical methods competitive immunochemistry - fast statim orientation determination of the most usual drugs of abuse, urine screening on diagnostic strip instrumental techniques - GC-MS or HPLC, serves mostly for consequent quantitative analysis after positive screening test screening imunoassay multifunctional (up to 9-zone) strip test, most frequently: amphetamine, barbiturates, benzodiazepines, cocaine, methamphetamine, morphine, phencydidine and tricyclic antidepressives (or their metabolites) 262 procedure: drug from urine (mAg) compete for binding sites with (usually) murine monoclonal antibody against it (Ab^), fixed on surface of microparticles, which are sorbed (not immobilised) in lower part of strip; in upper part of strip, there is immobilised other (second) antibody hb2 against murine immunoglobulins of Abj* after sinking of multi-strip into urine sample containing some of tested DOAs, the antibody on the start reacts and creates coloured immunocomplex [Ab^-mAg] and rises up urine without DO A. complex is not created and coloured microparticles with fixed antibody rise up and in the middle part reacts Abj* with on-there immobilised DOA/metabolite and creates there coloured immunocomplex [Ag-Abj*] as negative control urine with DOA. coloured immunocomplex [Ab^-mAg] created with sample rises and is captured at the end of strip, where it reacts with Ab2 and creates strongly coloured zone as positive test [Ab2.Ab1>|c-mAg]; antibody Abj* is in that case antigen for second fixed antibody Ab2 sensitivity. 10 - 100 ng/ml colloid gold (Au) microparticles are used, resulting colour of negative control and also positive test is red or blue (if the surface of the microparticle is blue) 263 glucose content: represents metabolism of saccharides determination: in serum, plasma and urine (most commonly determined analyte); reference interval (mM) in serum 3.9 - 6.1, in plasma 3.3 - 5.6 and in 24 h collected urine up to 1.39 chemical methods reaction with o-aminotoluene specific (also only galactose and mannose), supernatant of deproteinated sample in medium of glacial acetic acid, when o-aminotoluene (6 % o-toluidine in 80 % acetic acid containing 0.5 % of oxalic acid) condensates with Glu under elevated temperature into glycosylamine, stabilised by Schiff base, green product at ca 630 nm : agressive chemicals, necessity of boiling and sample deproteination enzymatic methods enzymes: hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), glucose oxidase (GOD), glucose dehydrogenase (GDH), peroxidase (POD) determination with GDH R-CH=0 + H20 + NAD+ £dh> R-COOH + NADH + H+ procedure: sample is mixed with working solution (phosphate buffer pH 7.6, GDH ca4.5 kU/l and NAD+ ca 2.2 mM), incubated for 7 min at 37 °C, absorbance increase at 340 n m 264 determination with H K and G6PD glucose + ATP ^> glucose-6-phosphate + ADP glucose-6-phosphate + NADP+ g6pd> g|Uconolacton-6-phosphate + NADPH + H+ procedure: sample with working solution (TRIS 50 mM pH 7.5, ATP 1 mM, NAD+ 2 mM, HK ca 3 kU/l and G6PD 2 kU/l) at 37 °C, after 5 min at 340 nm; HK is for glucose unspecific enzyme, specificity lies in consequent reaction determination by oxidative copulation with GOD and POD most practical R-CH=0 + 02 + H20 GQD> R-COOH + H202 R-NH2 + C6H5-OH + 2 H202 HQd> R-N=C6H4=0 + 4 H20 procedure: sample with working solution (phosphate ca 0.15 mM pH 8.0, GOD 10 kU/l, POD 1 kU/l, AAP 1 mM and 3-methylphenol 10 mM) at 37 °C, absorbance change at 500 nm in interval 30 - 90 sec or resulting colour after 15 min; second reaction in unspecific, is interfered by reductive compounds (vitamin C) special analysers personal glucometers for self-control of diabetics procedure: immobilised GOD and electrode system in dry state, indication by amperometry (so called Clark electrode): H202 Ht> 2 H+ + 02 + 2 e- or: glucose + 02 + R-Fe(II) hod> gluconate + R-Fe(III) 265 pyruvate content: product of lactate oxidation catalysed by LDH enzyme, determination: in blood only in small amount approx. 41 - 67 nM enzymatic methods enzyme: lactate dehydrogenase (LDH) pyruvate + NADH + H+ *^> L-lactate + NAD+ creatinine content: cellular product of muscular energetic metabolism of creatine determination: basic investigation of serum and urine (kidney function - creatinine clearance); amount is proportional to muscle mass size; reference interval for serum creatinine is under 115 pM chemical methods Jaffé reaction red-orange Janovsky complex (adduct of creatinine with picrate 1:1); unspecific, react also non-creatinine chromogens: proteins, glucose, ascorbic acid, guanidine, acetone, acetoacetate and pyruvate procedure: serum with working solution (picric acid 4.4 mM, NaOH 150 mM and Na2HP0413 mM), absorbance after 10 s or more precisely after 2 min at 492 nm 266 enzymatic methods determination through quinonimine dyes enzymes: creatininase (KRN), creatinase (KR), sarcosine oxidase (SOX), peroxidase (POD) creatinine + H20 %£&> creatine creatine + H20 ^> sarcosine + urea sarcosine + H20 + 02 §^x> glycine + HCHO + H202 2 H202 + 4-aminoantipyrine + phenol EQe> quinonimine dye + 4 H20 procedure: suitable derivatives of phenol - TBHB (2,4,6-tribromo-3-hydroxybenzoic acid) and EHSPT (N-ethyl-N-(2-hydroxy-3-sulphopropyl)-m-toluidine), absorbance at 550 nm triacylglycerols (TG) content: 95 % of all lipids stored in tissues are created by saturated fatty acids C12 to C18 determination: in serum (independent factor of ischemia of the heart muscle risk), reference interval lower than 1.7 mM chemical methods coloured product absorbing at 570 nm, or must be transformed by acetylacetone and ammonium acetate (Hantzch condensation) into yellow 3,5-diacetyl-l,4-dihydrolutidine, which might be determined by photometry or fluorimetry 267 described approaches maybe (after extraction of TG) described by these equations: TG + ROH/OH- -^ glycerol + 3R-COOH glycerol + I04--»HCHO + HCOOH + H20 + I03" HCHO + chromotropic acid + H2S04 -^ colour product OH O H03S S03H OH OH S03H H03S. x, ""HO.S .S03H OH OH or: 4 HCHO + NH4OH + CH3COCH2COCH3 -+ 3,5-diacetyl-l,4-dihydrolutidine + 5 H20 enzymatic methods combination with chemical method, use of created glycerol enzymes: glycerol kinase (GK), glycerol-3-phosphate dehydrogenase (G3PD), pyruvate kinase (PK), lactate dehydrogenase (LDH), diaphorase (DF), lipoprotein lipase (LPL), glycerophosphate oxidase (GPO), peroxidase (POD) 268 glycerol + ATP ^> glycerol-3-phosphate + ADP glycerol-3-phosphate + NAD+ g3pd> dihydroxyacetone phosphate + NADH + H+ ADP + PEP hk> ATP + pyruvate pyruvate + NADH + H+ ^*> lactate + NAD+ NADH + H+ + INT ^> red formazan INT + NAD+ INT - iodonitrotetrazolium violet, ca 500 nm combination with chemical methods too complex and not automatable determination through quinonimine dyes TG + 3 H20 ^> glycerol + 3 R-COOH glycerol + ATP ^> glycerol-3-phosphate + ADP glycerol-3-phosphate + 02 QEQ> dihydroxyacetone phosphate + H202 2 H202 + 4-aminoantipyrine + phenol EQ^> quinonimine dye + 4 H20 phenols: 4-chlorophenol, or 3,5-dihydroxybenzene sulphonic acid (DHBS), or N-ethyl-N-(3-sulphopropyl)-m-anisidine (ESPAS) determination through NADH TG + 3 H20 *^=> glycerol + 3 R-COOH glycerol + ATP ^> glycerol-3-phosphate + ADP glycerol-3-phosphate + NAD+ g3pd> dihydroxyacetone phosphate + NADH + H+ procedure: with only one solution, one-step and automatable 2ß< rheumatoid factor content: pentameric immunoglobulins IgM with specificity for Fe fragment of IgG, similar properties also within monomeric IgM, IgA and IgG determination: in serum, indication (up to 75 %) of all rheumatic disorders latex fixation test procedure: separation according to Cohn (extraction by set of ethanol solutions on rocks), fraction II is mixed with latex particles, on which y-globulin is passively sorbed, agglutination appears; sensitivity from 2 U/ml Rose-Waaler test haemagglutination test (ovine erythrocytes modified by tannin /strengthens cells/ covered by lapine antibodies against ovine erythrocytes); erythrocytes do agglutinate with sample containing human rheumatoid factor based on cross reaction between lapine and human IgG 270 C-reactive protein (CRP) content: belongs to so-called proteins of acute phase and to risk factors of ischemia of heart muscle determination: in serum, its concentration increases ca7 h after the inflammation and culminates during 1 to 3 days, when it may reach thousand of mg/l, reference interval is under ca 1.6 mg/l CRP may be determined by various immunochemical methods, most commonly by latex particle methods immunoturbidimetric determination DuREL (dual-radius enhanced latex) method - latex microparticles in two sizes with two types of monoclonal antibodies fixed with different affinity to CRP (large particles with higher affinity); at low analyte concentration larger particles react preferably, at higher concentrations also the smaller =^> sensitive, broad and linear measuring range CRP +Ab^[CRP-Ab] : turbidity at 340 nm 271 trypsin content: EC 3.4.21.4, serine proteinase, in duodenum determination: in plasma, in duodenal liquid, in faeces; test of exocrine pancreatic function disorder, reference interval approx. 150 ± 11 pg/l enzymatic methods immunochemically (namely RIA) photometry with chromogenic substrates L-TAPA + H20 in3£Bsin> N-a-tosyl-L-arginine + 4-nitroaniline yellow chromophore 4-nitroaniline; similar chromogenic substrate benzoyl-L-arginine-4-nitroanilid procedure: sample is diluted with physiological solution + buffer TRIS 200 mM pH 8 + substrate 1 mM, at 37 °C after 10 min absorbance at 405 nm 272 1 expectancy test (hCG) content: human chorionic gonadotropine (hCG), glycoprotein, dimer ä 40 kDa; hormone produced by placenta determination: in maternal blood and urine; since 8th day after conception, hCG level in urine during pregnancy increases dramatically, highest at 8th week, positive test at hCG content above 20 - 50 U/l latex agglutination test procedure: on support glass; in urine, hCG reacts with monoclonal antiserum (Ab, murine), and either inhibits following agglutination reaction after addition of latex particles with fixed hCG, or nor (reaction went on); in-parallel so-called positive control is ran hCG low content (cannot bind all antiserum) => agglutination happens, negative: hCG + Ab -> [hCG-Ab] + Ab Ab + hCG-latex -> [Ab-hCG-latex]^ hCG high content (all bound) => agglutination happens not, positive: hCG + Ab -> [hCG-Ab] [hCG-Ab] + hCG-latex -> no agglutination 273 diagnostics strip test with colloid gold strip contains in site of sample introduction (down) sorbed, but not fixed monoclonal antibody against hCG on colloid gold particles (Ab^Au), up (in the direction of diffusion) there are two zones with immobilised antibodies; first zone contains fixed secondary antibody for hCG(Ab2b), second zone contains fixed antibody for Abx (Ab3b) hCG high content hCG reacts with primary antibody into complex, diffuses up and is caught in the first zone with fixed Ab2 as {Ab2-hCG-AuAbl} sandwich) red zone appears í positive reaction) hCG low content no complex, Abl diffuses up, and is caught in the second zone) there appears red zone of colloid gold (negative reaction), serves as positive control : instead of colloid gold e.g. organic dye 274 strip with antibody labelled with enzyme : similar to colloid gold : primary hCG antibody is labelled with suitable enzyme (Ab^); in a location of fixed (Ab2b) is in reaction zone suitable substrate and auxiliary compounds hCG high content complex [Ab^-Ag] is created, which diffuses and is caught on Ab2b => [Ab^-Ag]- Ab2b; enzyme catalyses decomposition of colourless substrate and zone turns coloured hCG low content no complex, labelled primary antibody is caught in control zone by thirdantibody(Ab3b) [Ab1*-Ab3b]; second zone also contains enzyme substrate - coloured zone substrates: : for enzyme POD - TMB (in acidic medium) : blue colour : for enzyme ALP - Phenolphthalein- or thymolphthalein phosphate (alkali medium) : red to blue colour quantification ofhCG by ELISA sandwich technique; second Ab is a conjugate with POD, substrate ABTS 275 Mycobacterium tuberculosis content: Mycobacterium is acido-resistant bacillus, pathogenic (tuberculosis) M. tuberculosis and M. bovis, atypical mycobacteria, e.g. M. Kansas/' and M. avireni and etiological agent of leprosy M. leprae, less than 10 bacilli are already infectious determination: sputum, morning sample, ca 5 - 10 ml 3 days one after one, sometimes also liquor, purulence, biopsy, urine; not stainable according to Gram cultivation proof procedure: decontamination (non-specific microflora) NaOH 1 M in presence of laurylsulphate, HCl neutralisation; medium inoculation {egg with salts, asparagine, glycerine, stark and malachite green; or fluid containing glutamate, L-alanine, casein hydrolysate, bovine serum etc.), incubation at 37 °C, recorded after 1, 3, 6 and 9 weeks (excluding contaminated media); within positive result it proceeds with detection of caught strains and starts with sensitivity test (resistance) towards drugs; for distinguishing M. tuberculosis and M. bovis the ability to produce niacin is evaluated, by reduction of nitrates to nitrites, sensitivity towards thiophene-2-carbonic acid hydrazide (so called TCH-test) and hydrolysis by Tween 80; M. tuberculosis has, in contrast to M. bovis, all listed tests positive PCR proof HIV (AIDS) content: body fluids, virus of human immunodeficiency, retrovirus determination: in serum, immunochemical approaches (ELISA) 276